Molecular Biology, Epidemiology, and Pathogenesis of ProgressiveMultifocal Leukoencephalopathy, the JC Virus-Induced DemyelinatingDisease of the Human Brain
Michael W. Ferenczy,a Leslie J. Marshall,a Christian D. S. Nelson,b Walter J. Atwood,b Avindra Nath,c Kamel Khalili,d andEugene O. Majora
Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USAa;Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USAb; Section of Infections of the Nervous System, NationalInstitute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USAc; and Department of Neuroscience, Center for Neurovirology,Temple University School of Medicine, Philadelphia, Pennsylvania, USAd
Historical Association of Immunological Risk Factors andJCV with PML
Before the AIDS pandemic and the use of immunomodulatorytherapy, progressive multifocal leukoencephalopathy (PML)
was an extremely rare disease, associated primarily with underly-ing neoplastic conditions causing a defect in immune function(20, 419). Interestingly, PML was associated mainly with B celllymphoproliferative disorders (57, 198), which have been hypoth-esized to lead to the spread of virus from potential sites of latencyto the brain. Accounts of potential cases of PML can be traced backas far as 1930 (29, 85, 181, 419, 537). The first case of demyelinat-ing disease described with the term PML was found in a patient
with chronic lymphocytic leukemia (CLL) and Hodgkin’s lym-phoma in 1958 (20). These cases are all consistent with the pathol-ogy of PML, including the development of multiple white matterplaques in the brain stem, basal ganglia and thalamus, cerebralhemispheres, and cerebellum.
A viral cause of PML was proposed in 1959 due to observationsof inclusion bodies in the nuclei of damaged oligodendrocytes
(70) and the hypothesis that the distribution of lesions could beexplained by an atypical viral infection (419). The nuclei of cellswith inclusion bodies were found by electron microscopy to con-tain particles similar to known polyomaviruses (202, 559, 560).The etiological agent of PML was not isolated until 1971, when thevirus was isolated from a mixed culture of glial cells following a“blind” passage (380) and named JC virus (JCV), after the initialsof the patient. More recently, JCV has been termed JC polyoma-virus (JCPyV), but this review will maintain the more commonnomenclature of “JCV.”
JCV was found to be a nonenveloped icosahedron of 40 nmdiameter, which, unlike simian virus 40 (SV40), could cause hem-agglutination (HA) of human type 0 erythrocytes (377), whichprovided means to perform seroepidemiological studies. Datafrom these studies indicated that JCV was found globally (58), thatseroconversion of a large percentage of the population occurredbefore adulthood (431, 522), and that healthy people, includingpregnant women, produced immunoglobulin G (IgG) againstJCV (17, 99). Therefore, PML was likely to be caused by reactiva-tion of a latent infection (57, 379, 522). For a full review of thehistorical association of JCV and PML, see reference 301 and ref-erences therein.
PML ceased to be a rare disease after HIV became widespreadin the human population. Estimates of the occurrence of PML inAIDS patients range from 3 to 5% (299). The incidence of PML inAIDS patients is significantly greater than that in persons withother underlying causes of immunosuppression (299). Notably,PML incidence has decreased less significantly than other oppor-tunistic infections since the advent of highly active antiretroviraltherapy (HAART) (103, 546). It is unclear why PML occurs morefrequently in AIDS patients than in those with other underlyingcauses of immunosuppression, although some causes may includechanges in immune cell trafficking, blood-brain barrier (BBB)permeability and cytokine secretion, interaction between viralproteins in coinfected cells, and damage to the brain caused byneuronal HIV infection. HIV likely affects both the immune sys-tem and the local cellular environment in ways that increase therisk of progression to PML.
The development of PML as a side effect of immunomodula-tory therapy is a growing concern, with reports of fatal PML casesin patients treated with natalizumab (Tysabri) for multiple scle-rosis (MS) and Crohn’s disease, with rituximab (Rituxan) formultiple sclerosis, non-Hodgkin’s lymphoma, rheumatoid arthri-tis, autoimmune hematological disorders, myasthenia gravis, sys-temic lupus erythematosus (SLE), and B cell lymphoma, withefalizumab (Raptiva) for plaque psoriasis, with infliximab (Remi-cade) for psoriasis, Crohn’s disease, ankylosing spondylitis, psori-atic arthritis, rheumatoid arthritis, and ulcerative colitis, and withmycophenolate mofetil (Cellcept) for suppression of organ trans-plant rejection (315). These therapies target immune cells, inhib-iting their biological function. The risk of PML during natali-zumab treatment rises as treatment progresses. The true overallincidence of PML due to natalizumab therapy is currently esti-mated at 3.85 per 1,000 patients (available for prescribing physi-cians at https://medinfo.biogenidec.com) and approximately 1per 500 efalizumab patients. Prescription labeling for mycophe-nolate mofetil contains a warning of PML risk. Natalizumab andrituximab contain FDA-mandated “black box warnings” becauseof the risk of PML (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/), while efalizumab has been voluntarily withdrawn
from the market because of concerns about the frequency of oc-currence of PML (299). Although these therapies are effective fortheir intended use, the incidence of PML, a deadly disease with noapproved treatment, limits their use. Thus, continued researchand a more thorough understanding of JCV biology, epidemiol-ogy, and pathology are of continued and increasing importance.
Viral Structure, Proteins, and Genome
JCV, like all polyomaviruses, is a nonenveloped, T � 7 icosahedralvirus with a closed circular, supercoiled, double-stranded DNAgenome. The capsid is composed of three viral structural proteins,VP1, VP2, and VP3, with VP1 being the major constituent. Thereare 72 pentamers, each composed of five VP1 molecules and onemolecule of either VP2 or VP3 (302). Only VP1 is exposed on thesurface of the capsid, and it thus determines receptor specificity.Polyomavirus DNA is nucleosomal in structure, with approxi-mately 25 nucleosomes composed of viral DNA and host cell his-tones contained in each minichromosome, as determined forSV40 (12, 264, 333). The viral particle does not contain linkerhistones, but the genome acquires them after entry into the hostcell.
The prototype JCV genome (Fig. 1) is 5,130 bp (154), althoughindividual variants differ in length, due to alterations in their non-coding regions (see Fig. 3). The genome encodes 6 major viralproteins (large T and small t antigens, VP1, VP2, VP3, and agno-protein) as well as several splice variants of T antigen. Early- and
FIG 1 Schematic diagram of the JCV genome. The circular Mad-1 genome is5,130 bp and codes for 9 proteins: large T antigen (T), small t antigen (t),T=135, T=136, T=165, VP1, VP2, VP3, and agnoprotein. At the top is the NCCR,composed of the origin of replication (ORI) and two 98-bp repeats. Map unitsare denoted in the center circle. The early open reading frame (ORF) proceedsfrom the NCCR in the counterclockwise orientation and the late ORF in theclockwise direction. Proteins in the same reading frame are depicted in thesame color. Start and stop sites are indicated. All transcripts are polyadenylated[(A)n].
late-transcribing sides of the genome are physically separated by anoncoding control region (NCCR), often called the hypervariableregulatory region (HVRR) or regulatory region (RR), and aretranscribed in opposite directions from opposite strands of DNA.The early side of the genome, which is transcribed before DNAreplication begins, is composed of large T antigen and small tantigen genes, as well as the splice variants T=135, T=136, and T=165.The late side of the genome is transcribed concomitant with DNAreplication and encodes the three viral structural proteins, VP1,VP2, and VP3, as well as the accessory agnoprotein.
Large T antigen and its variants are multifunctional, interactingwith both host and viral proteins as well as with both host and viralDNAs. The T proteins are involved in driving the host cell towardS phase for viral replication, regulating transcription from the hostand viral genomes, and directly participating in viral DNA repli-cation. The agnoprotein is less well studied but also appears to bemultifunctional, and highly varied functions have been attributedto it (reviewed in reference 238), ranging from viral transcrip-tional regulation to inhibition of host DNA repair to functioningas a viroporin (478).
VIRUS RECEPTORS AND HOST CELL INVASION
Polyomaviruses all display restricted species and cell type specific-ities for lytic infection. The strict species specificity of JCV forhumans has confounded the development of animal models forPML. In vivo, JCV infection is likely restricted to kidney epithelialcells, tonsillar stromal cells, bone marrow-derived cell lineages,oligodendrocytes, and astrocytes (22, 201, 300, 301, 341, 342,494). The virus is thought to establish low-level persistent or latentinfections in the kidney and in bone marrow-derived cells largelydue to inefficient viral replication in these cell types. Once in thecentral nervous system (CNS), the virus replicates vigorously inoligodendrocytes, leading to the demyelinating disease PML. Thecell type-specific tropism of JCV observed in vivo is mirrored invitro, with virus productively infecting bone marrow-derivedcells, tonsillar stromal cells, and macroglia (142, 303, 306, 307,344). Virus replication is maximal in primary human fetal glial cellcultures and in some human glial cell lines (303), as well as in othercell lines expressing SV40 large T antigen, such as COS-7 cells(185, 303). However, it has been our experience that JCV does notmultiply well in COS-7 cells compared with the human SVG cellline, which was established with the same SV40 vector as the COScell lines.
The major tropism of JCV for human glial cells is not fullyunderstood, but multiple factors are likely responsible for con-tributing to robust viral replication in this cell type. Host cell- andspecies-specific transcription and replication factors contributesignificantly to the restricted specificity displayed by JCV andother members of the genus (142, 292, 480). In addition, virus-receptor interactions contribute to viral tropism and spread. Thiswas first shown for JCV using a JCV/SV40 hybrid virus that con-tained the NCCR and T antigen-coding genes of SV40 as well asthe JCV capsid-coding genes (76). The hybrid virus induced anSV40-like cytopathic effect in human glial cells and hemaggluti-nated human type O red blood cells similarly to JCV (76).
Virus Receptors
In all polyomaviruses studied to date, initial binding to host cellsinvolves an interaction with negatively charged sialic acid-con-taining receptors. The early binding and entry events in the related
polyomaviruses SV40 and murine polyomavirus (mPyV) havebeen extensively studied and contribute to our understanding ofJCV pathogenicity. The monosialylated ganglioside GM1 is themajor receptor for SV40 (26, 63, 499). SV40 has also been shownto use the class I major histocompatibility complex protein 1 as areceptor in some cases (54, 366, 468). Initially, it was thought thatSV40 was not able to attach to sialic acids, as neuraminidase treat-ment of host cells did not reduce infectivity, although it was latershown that neuraminidase fails to cleave sialic acid off GM1 (88,337). This interaction between SV40 and GM1 is extremely spe-cific, as SV40 preferentially binds to N-glycolyl GM1 over N-acetylGM1, which differ by a single hydroxyl group (63). Additionally,although a single VP1 molecule binds GM1 weakly, the multiva-lent viral capsid is expected to bind GM1 molecules on the plasmamembrane with an affinity that is several orders of magnitudegreater (362).
Early studies with mPyV demonstrated that the virus binds to�2,3- and �2,6-linked sialic acids on host cells (61, 62, 149, 383).Subsequently, the mPyV was shown to bind to sialic acids presenton the ganglioside receptors GD1a and GT1b (461, 499). Al-though mPyV utilizes gangliosides for productive infection, thevirus will bind to sialic acids present on glycoproteins (397). Bind-ing of mPyV to cell surface glycoproteins targeted the virus to anonproductive pathway for degradation, suggesting that theseglycoproteins represent nonproductive pseudoreceptors. The spe-cific interactions between mPyV and sialic acids also play a criticalrole in pathogenicity. Early studies demonstrated that while bothsmall- and large-plaque variants of mPyV were able to bind to�2,3-linked sialic acids, small-plaque variants were additionallyable to bind �2,6-linked sialic acids due to a single point mutationin VP1 (62, 148, 469, 470). As a result, large-plaque variants boundto cells less tightly. This reduced binding also increased pathogen-esis of mPyV in mice, indicating that the reduced binding to sialicacids increased viral spread (123, 148).
JCV also requires sialic acid to infect cells and has been reportedto utilize both �2,3- and �2,6-linked sialic acids to infect permis-sive glial cells (125, 284). After stripping glial cells of �2,3- and�2,6-linked sialic acids, each linkage was reconstituted using link-age-specific sialyltransferases. Restoring either �2,3- or �2,6-linked sialic acids to cells rescued infection, indicating that both�2,3- and �2,6-linked sialic acids play an important role in infec-tion (125). The sialyltransferases used were also specific for addingcarbohydrate to N-linked glycoproteins, and as infection was fullyrestored, it was suggested that an N-linked glycoprotein contain-ing either �2,3- or �2,6-linked sialic acid was sufficient for virusinfection.
Recently, a receptor moiety used by JCV for productive infec-tion has been identified. Using glycan arrays, a recombinant JCVVP1 pentamer has been shown to bind specifically to lactoseriestetrasaccharide C (LSTc), which contains a terminal �2,6-linkedsialic acid (361). This unusual molecule adopts an “L” shape, andJCV specifically binds this molecule by interacting with not onlythe terminal �2,6-linked sialic acid but also the adjacent GlcNAcsugar. Comparing the crystal structures of SV40 in complex with aportion of GM1 and of JCV in complex with LSTc reveals that the�2,3 versus �2,6 linkages and the favorable interactions betweenJCV and GlcNAc largely determine binding specificity. Sialylpara-globoside, which is identical to LSTc except for its terminal �2,3-linked sialic acid, does not bind JCV. Although �2,3-linked sialicacids were present on the glycan array, none demonstrated appre-
Molecular Biology, Epidemiology, and Pathogenesis of PML
ciable binding to the VP1 pentamer. When the multivalent JCVvirus-like particles (VLPs) were used, low binding to some �2,3-sialic acids, including the gangliosides GM2 and GM1, was seen.Therefore, it appears likely that JCV primarily uses �2,6-linkedsialic acids to bind to cells, with the possibility that the weak in-teractions between JCV and �2,3-linked sialic acids play a limitedrole in infection. It is currently unknown onto what receptor mol-ecule the LSTc moiety is attached.
Viral DNA recovered from cerebrospinal fluid specimens (CSF)from patients with PML has been shown to code for amino acidsubstitutions of several VP1 residues responsible for binding tosialic acids (477). These mutations are predicted to reduce bindingto sialic acids, although it is unclear whether these mutations willconfer a selective advantage in patients, as similar mutationsclearly reduce infectivity of JCV in tissue culture (362). Alterna-tively, these mutations may result in a selective advantage similarto that seen in mPyV by preventing nonproductive adsorption tocells that are not permissive for JCV infection. However, thesemutations appear to block binding to both �2,3- and �2,6-linkedsialic acids, and JCV VLPs containing these mutations show areduced hemagglutination ability and a decreased ability to bindgangliosides (172, 361). Interestingly, these mutations were recov-ered only in the CSF and bloodstream and not in the same pa-tient’s urine. As only a single genotype of virus was recovered ineach patient, it appears that these mutations arose from positiveselection of a single JCV population (413). It is currently unclearwhether these mutations arise in the CNS of PML patients ororiginate in peripheral sites before trafficking to the brain. Furtherstudies will be necessary to determine the effects of these muta-tions on JCV pathogenesis.
In addition to using sialic acid as a receptor, JCV has beenshown to require the serotonin receptor, 5HT2AR, to infect glialcells (130). The initial observations were centered around the factthat antipsychotic drugs such as chlorpromazine (Thorazine) andclozapine potently inhibit virus infection (30). As these drugs an-tagonize both serotonin and dopamine receptors, it was hypoth-esized that one or both of these neurotransmitter receptors mightfunction as a virus receptor on glial cells. Subsequent studies usingdrugs that specifically antagonized dopamine or serotonin recep-tors suggested that the 5HT2A subtype of the serotonin receptorfunctioned as a JCV receptor (130, 373). It was then shown thatexpression of the 5HT2A receptor was sufficient to allow viral entryto cells lacking this receptor (HeLa and HEK293A) and that anti-bodies to the 5HT2AR blocked infection (130, 296). These datawhen taken together strongly support a role for 5HT2AR in JCVinfection of glial cells. In vivo data demonstrate increased expres-sion of the 5HT2A receptor in and around PML lesions, furthersupporting a critical role for this receptor in the pathogenesis ofPML (unpublished observations). Interestingly, infection of5HT2AR-expressing cells remains neuraminidase sensitive, con-firming a prominent role for cellular carbohydrates in infection(unpublished observations). The 5HT2AR protein contains severalpotential glycosylation sites, but it is unclear whether LSTc orother sialic acid moieties responsible for infection reside on thisprotein. Elimination of glycosylation sites on 5HT2AR also pre-vents receptor expression of the cell surface, preventing a clearconsensus on the need for sialic acids on 5HT2A for infection(296). Alternatively, LSTc may instead be present on other sialicacid-containing molecules. One group has found that JCV caninfect human brain microvascular endothelial cells that lack the
5HT2A receptor (75). Infection in their system was very inefficient,but the results indicate that virus infection can proceed by alter-native mechanisms on some cell types. Additionally, gangliosidesmay play a role in JCV infection, and the ganglioside GT1b hasbeen reported to function as a receptor for JCV (248).
Virus Entry
Unlike mPyV, SV40, or BK virus (BKV), JCV enters cells by clath-rin-dependent endocytosis (389; reviewed in reference 500) (Fig.2). Pharmacological inhibitors and expression of dominant nega-tive proteins directed at clathrin-dependent endocytosis inhibitvirus infection, whereas inhibition of caveola-dependent endocy-tosis has no effect on replication (389). Consistent with clathrin-mediated endocytosis, JCV traffics from clathrin-coated pits toRab5-positive early endosomes, a step that is blocked by dominantnegative Eps15 mutants (398) (Fig. 2). Additionally, JCV colocal-izes with green fluorescent protein (GFP)-labeled Rab5, and ex-pression of dominant negative inhibitors to Rab5 prevent infec-tion. Subsequently, JCV colocalizes with cholera toxin B, a markerfor lipid-mediated endocytosis used by SV40 and mPyV, in com-partments that are most likely caveolin-1-positive late endosomes(traditionally referred to as caveosomes) (134, 140, 399). Surpris-ingly, JCV does not colocalize with markers for Rab7 late endo-somes, and expression of the dominant negative form of Rab7does not inhibit infection. This suggests that trafficking to Rab7-positive late endosomes is not necessary for infection and thatvirions may leave the normal endocytic pathway from Rab5-pos-itive endosomes or Rab5- and Rab7-positive maturing endo-somes. At 12 to 16 h postinfection, JCV colocalizes with the endo-plasmic reticulum (ER) protein calregulin, suggesting that JCVtraffics to the ER for productive infection. Treatment of cells withbrefeldin A, which inhibits COP1-mediated ER trafficking, po-tently inhibits infection, which further suggests that trafficking ofJCV to the ER is a critical step in infection (399).
There is probably much to be learned from SV40 and mPyVthat has not been determined for JCV. Delivery of polyomavirusesto the ER is a critical step in infection. In the ER, both mPyV andSV40 interact with components of the host protein folding andquality control machinery for productive infection. It has beendemonstrated that mPyV interacts with the protein disulfideisomerase (PDI) family of proteins PDI, ERP57, ERP72, andERP29 (297, 521). The 72 VP1 pentamers that associate to formthe viral capsid are stabilized by interactions between the C-ter-minal residues of neighboring pentamers, disulfide bonds formedbetween neighboring pentamers, and calcium ions coordinated bythe viral capsid (276). Interaction with the PDI family of proteins,which can isomerize and disrupt disulfide bonds, is able to disruptinterpentameric binding and capsid stability. ERp29 contains onlya single catalytic site yet retains its isomerization ability (187).ERp29 interacts with mPyV, and this interaction results in expo-sure of the C-terminal domain of VP1 (297, 401). Additionally,the previously internalized minor capsid proteins, VP2 and VP3,are exposed as a result of interaction with ERp29. This results in analtered virion that is capable of binding and penetrating lipid bi-layers (297, 400). Since the exposure of the C-terminal domain ofVP1 requires disulfide bond disruption, additional PDI proteinsappear to be necessary for infection by mPyV. Recently, PDI andERp57 have been identified as acting in a cooperative fashion withERp29 to reduce and isomerize the mPyV interpentameric disul-fide bonds (521).
In related work, similar ER-associated factors were found toplay critical roles in SV40 disassembly (443). Inhibition of en-dogenous cellular PDI and ERp57 by small interfering RNA(siRNA) reduced SV40 infectivity. Unlike the case for mPyV,knockdown of ERp29 had no effect on SV40 infection. Addi-tionally, in vitro interaction between ERp57 and SV40 uncou-pled the 12 five-coordinated VP1 pentamers of SV40. It is likelythat in the high-calcium environment of the ER, these pentam-ers remained attached to the virion. However, upon exit fromthe ER, the low-calcium environment of the cytosol allows forrelease of these pentamers.
A second critical step in the ER is the translocation of the virionacross the ER membrane. As a result of recent studies, it appearslikely that polyomaviruses utilize the ER-associated degradation(ERAD) pathway to traffic through a retrotranslocation pore andgain entry to the cytoplasm. Using short hairpin RNAs (shRNAs),Derlin-2 was identified as playing a critical role in mPyV infection.Derlin-2 is a protein involved in the transport of misfolded pro-teins out of the ER and into the cytoplasm to be degraded (277).Subsequently, Derlin-1 and SelL1, two proteins involved in theERAD pathway, were found to be important in SV40 infection(443). These interactions are predicted to facilitate the release of apartially disassembled and destabilized virion into the cytosol. ForSV40, it has recently been demonstrated that intact virions pene-trate the ER membrane during retrotranslocation to the cytosol
(210). This suggests that either the retrotranslocation pore used bySV40 is large enough to accommodate a 40-nm virion or thehydrophobic capsid residues exposed in the ER facilitate per-foration of the ER membrane and allow an intact virion toenter the cytosol.
After retrotranslocation, the low calcium concentrations en-countered by the virus in the cytosol further disrupt capsid stabil-ity. This destabilization is believed to result in shedding of a num-ber of the previously attached viral pentamers (443). As a result,nuclear localization signals are exposed, resulting in transport ofthis partially disassembled virion to the nucleus through the nu-clear pore (212, 226, 354, 548). It is likely that some combinationof VP1, VP2, and VP3 remains associated with the viral genomeduring nuclear entry, as minichromosomes that contain the viralgenome and nucleosomes do not efficiently enter the nucleuswhen microinjected into cells (354).
Thus, it is becoming increasingly clear that polyomaviruses uti-lize a complicated and novel pathway among viruses to ultimatelygain access to the host cell nucleus. JCV binds sialic acids similarlyto other polyomaviruses yet enters cells by clathrin-mediated en-docytosis, suggesting that gangliosides may not play a role in JCVentry. Further experiments will be necessary to determine whichreceptor molecules contain the sialic acids to which JCV binds forproductive infection. It will also be important to determine the
FIG 2 Representation of the early events involved in JCV infection. JCV (indicated by green viral capsids with supercoiled circular DNA) initially binds tocarbohydrate receptors (likely �2,6-linked sialic acid) on the cell surface. The sialic acid could be attached to the G-protein-coupled 7-transmembrane receptorfor serotonin (5HT2AR) or to another cell surface glycoprotein or glycolipid. The virus is then internalized into clathrin-coated pits and sorted into the earlyendosome. JCV colocalizes with cholera toxin, most likely in maturing or late endosomes. The virus then traffics to the to the ER by analogy with otherpolyomaviruses. It is likely that JCV interacts with PDI and ERAD proteins similar to those that interact with SV40 and mPyV in the ER. This is expected to causeconformational changes to the JCV virion, denoted by red shading of the capsid, and retrotranslocation of the virion into the cytoplasm. From there, it is likelythat JCV enters the nucleus through the nuclear pore.
Molecular Biology, Epidemiology, and Pathogenesis of PML
similarities between JCV, mPyV, and SV40 in the later steps ofvirus entry and trafficking.
PATHOGENESIS AND MOLECULAR REGULATION OF JCVINFECTION
Cell Type Specificity of JCV
Unlike other polyomaviruses such as BKV and SV40, JCV shows amore restricted host cell range, which has made biochemical andmolecular studies difficult. The viral T antigen interacts specifi-cally with human DNA polymerase, restricting the host range inwhich JCV can replicate (142). Originally, JCV isolates were ableto be grown only in human brain cells (540, 541). In order tofacilitate biochemical studies of the virus, several other culturemodels were developed. SVG cells are T antigen-dependent, im-mortalized human fetal brain cells derived by transducing a het-erogeneous culture of human fetal brain cells with a nonreplicat-ing, origin-defective SV40 vector that expresses the SV40 Tantigen (303). These cells allowed for the growth of JCV stocksand the study of viral gene expression and replication. Later, it wasdemonstrated that human fetal astrocytes in culture could sup-port JCV replication, raising the possibility that JCV could repli-cate in cells other than oligodendrocytes (307).
Several other cell lines that support JCV replication have beenestablished. A JCV-induced owl monkey glioblastoma cell linewhich spontaneously produced the Mad-4 variant of JCV was es-tablished in tissue culture (308). Transformed Rat2 cells were es-tablished to study JCV-induced transformation in culture (497),and the IMR-32 human neuroblastoma cell line was determinedto support JCV replication, which made it useful for propagatingvirus (6). JCV produced by persistently infected IMR-32 cells be-came cell culture adapted for growth in these cells (368, 372).Several other cell lines were established by fusing primary humanfetal astrocytes with the glioblastoma cell line U-87MG (441). Al-ternatively, a pseudovirus system was created using SV40 large Tantigen-transformed COS-7 cells expressing JCV structural pro-teins, which produced virus-like particles lacking viral genomes(451).
More recently, cultures of human fetal brain progenitor-de-rived astrocytes (PDAs) or progenitor-derived neurons (PDNs)derived from human fetal neural progenitor cells have been usedto study viral gene expression, replication, and growth in specificglial cell types (334). These cells allowed further study of the mo-lecular regulation of JCV by cell-specific factors in a more purepopulation of cells.
Although pathology of JCV occurs in tissues of the central ner-vous system, seroepidemiological studies show that more thanhalf the global population is either transiently or latently infectedwith JCV (378), although there is heterogeneity among popula-tions, even in nonindustrialized areas (305). Additionally, the per-centage of seropositive individuals increases with age (247), and10 to 30% of people shed JCV in the urine (299). Because directinfection of brain tissue is unlikely and would be exceedingly rare,dissemination by some more common route is probable. Furtherinquiry led to the observation that cells of the immune system,including hematopoietic progenitor cells and B lymphocytes, aswell as tonsillar stromal cells and Schwan cells are susceptible to JCvirus (19, 341, 342, 345). Additionally, it was found that JCV mayremain latent in immune cells of the bone marrow (198, 201, 322,484).
Despite the limited range of species and cell types permissive forJCV replication, JCV is ultimately a very successful pathogen, asillustrated by its wide dissemination but rare pathogenesis. Thissuccess is attributable to the tight regulation of the viral life cycle.The differential ability of JCV to bind, enter, transcribe gene prod-ucts, replicate, and ultimately produce more infectious viral par-ticles in various types of human cells is essential to the regulationand life cycle of the virus. The following sections discuss theseaspects of the viral life cycle, with emphasis on stages that regulatethe virus’ ability to complete its life cycle in various cells.
Analysis of the Viral Genome
Similar to the case for all other polyomaviruses, the JCV genome isa closed circular supercoiled chromosome that is composed of“early” and “late” genes that are separated by the noncoding con-trol region (NCCR), which contains the origin of replication(ORI), promoter, and enhancer elements (154) (Fig. 1 and 3). Theearly region is expressed de novo after infection and before DNAreplication and is on the ORI-proximal side of the NCCR. Lategenes are optimally expressed concurrently with or after DNAreplication and are found on the ORI-distal side of the NCCR. Thepolyomavirus NCCRs are the most variable portions of the viralgenome within a single virus as well as across genera of viruses(143, 151, 154, 267, 411, 446, 551).
The early-region sequences are transcribed counterclockwisefrom the NCCR and encode five proteins: the large T and small tantigens and splice variants called T=135, T=136, and T=165 (153,395) (Fig. 1). The JCV large T antigen is a 688-amino-acid non-structural, multifunctional protein that regulates viral early genetranscription and is thus autoregulatory. Large T antigen also reg-ulates the switch from early to late viral transcription, as well asviral DNA replication. Large T antigen interacts with a number ofcellular proteins to support transcription and replication of theviral genome, and upon unregulated overexpression, it inducescellular malignant transformation. The 172-amino-acid small tantigen shares the open reading frame (ORF) start site of nucleo-tide 5013 (339), and thus its 5= end, with large T, but it is differ-entially spliced (154) and contains a translation termination signalat nucleotide 4495 (339), as opposed to position 2603 for large Tantigen (154), which results in different 3= ends of small t and largeT antigens. Small t antigen and the other T antigen splice variantsalso perform multiple functions to drive infected cells toward Sphase and may contribute to gene expression and PML progres-sion to various extents in the presence of different underlyingdisorders (211).
The late region is transcribed clockwise from the oppositestrand of the genome, and is composed of the coding sequencesfor four proteins (154) (Fig. 1). The smallest protein, agnoprotein,has an open reading frame that codes for 71 amino acids, begin-ning at nucleotide 277 and terminating at nucleotide 492. Theagnoprotein is not well understood but has been proposed to be aviroporin (478), as well as to interact with large T antigen to de-crease viral DNA replication. It has also been demonstrated tointeract with transcriptional activators and repressors to controlgene activation as well as influence DNA repair pathways (101,238, 437). The other three open reading frames code for the viralstructural proteins VP1, VP2, and VP3. The open reading framesfor the structural proteins overlap. The 354-amino-acid majorcapsid protein VP1 is found at the 3= end of the late region (nu-cleotides 1469 to 2533), is the major structural protein, and func-
tions in cellular binding and entry. The ORFs for the minor capsidproteins, VP2 and VP3, are found between the 3= terminus of theagnoprotein ORF and the 5= end of VP1. VP2 is a 344-amino-acidprotein with an ORF between nucleotides 526 and 1560. VP3 iscomposed of the C-terminal 225 amino acids of VP2, with itscoding region starting at nucleotide 883 and sharing the 3= termi-nus of VP2 (154, 301).
The NCCR lies between the early and late coding sequences and
contains the origin of replication (152, 154, 339). It contains ex-tensive regions of homology with the polyomaviruses SV40 andBK virus, including two of the three T antigen binding sites foundin SV40 (152), as well as a unique non-B DNA tertiary structure(15). The NCCR is thought to be the main determinant of cell typespecificity and is composed of fairly well-conserved flanking re-gions that border the transcription start sites of the early and latecoding regions, as well as a central region containing numerous
FIG 3 DNA sequence block representation of the noncoding control regions (NCCRs) of selected viral variants, showing DNA sequence block arrangements ofthe NCCRs of the prototype variant Mad-1 (A), Mad-8, which has similarity to and is illustrative of the majority of NCCR arrangements found in PML tissue (B),and the archetype variant CY, which is commonly found in urine of healthy individuals (C). Changes in NCCR sequence result in changes in transcription factorbinding sites, which affect tissue specificity and activity of viral transcription and DNA replication. Changes in transcription factor binding are complex and differmarkedly between variants of JCV found in PML patients. Depicted are the DNA sequence blocks known to make up JCV NCCR rearrangements, with thedirection of transcription indicated by arrows. The numbering scheme is that of Frisque et al. (154). The origin of replication is denoted “ORI.” The lowercaseletters “a,” “b,” “c,” “d,” “e,” and “f” indicate sequence blocks. The italicized “b” in the Mad-8 sequence represents a deletion of part of the “b” sequence. Red linesin Mad-8 represent an insertion of a single extra adenine between sequence blocks. The locations of TATA boxes, as well as binding sites for JCV large T antigen(black box with “T”) and sequences similar to the HIV tat-responsive element, known as the transactivation response element (TAR), are above the DNAsequence representation. Known binding sites for cellular proteins are underneath the DNA sequence blocks, and Most sites have been determined in Mad-1,with sites in the “b” and “d” sequence blocks determined in archetype. Sites in Mad-8 and “a,” “c,” and “e” sites of archetype have not been experimentally shownand have been determined through sequence similarity to known, experimentally determined binding sites in Mad-1, Mad-4, or archetype variants. Sites foundonly in Mad-1 are shown in yellow. Binding sites in both Mad-1 and Mad-8 are orange, those found in both Mad-8 and archetype are green, and those found onlyin archetype are red. Abbreviations are as follows: 1, Sp1; 2, SF2/ASF; 4, NFAT4; A, AP-1 (c-jun); B, Bag-1; C, C/EBP�; D, DDX-1; G, GF-1/S�BP-2; H, HIF-1�;i, GBP-i; K, NF-�B; L, LCP-1; N, NFI; O, Tst-1/Oct-6/SCIP; P, Pur� and YB-1; S, Spi-B. References can be found in the text.
Molecular Biology, Epidemiology, and Pathogenesis of PML
transcription factor binding sites. The early-proximal side of theNCCR contains the preorigin and origin of replication. TheNCCR varies greatly between isolates from PML patients. In ad-dition, a sequence, known as archetype or CY, has been isolatedfrom urine specimens from both PML patients and healthy peoplebut is rarely found in PML brain tissue. The original sequenceisolated from a PML patient is known as Mad-1, as it was isolatedat the University of Wisconsin—Madison. Subsequent isolateshave been named with various designations, often depending ontheir associated immunosuppression or locations of isolation,with Madison isolates being known as Mad-1, Mad-2, and soforth.
Naturally occurring variants. The NCCR from the original iso-late of JCV, Mad-1, contains an enhancer element that exists as a98-bp direct tandem repeat and therefore contains duplicateTATA boxes, which can position mRNA start sites (165, 167), aswell as multiple transcription factor binding sites (152). The tan-dem repeat structure of the Mad-1 NCCR variant has been termedthe “prototype” JCV NCCR sequence and is composed of threeblocks of sequence, named “a,” “c,” and “e,” with the TATA boxfound in “a” (Fig. 3A). Many studies have demonstrated that theTATA box(es) contained in the 98-bp direct repeat structure isessential for transcription of early and late viral genes (95, 230,231, 234, 253, 503). Numerous NCCR variants containing a tan-dem repeat-like structure have been isolated from tissues of pa-tients with PML (317). Although Mad-1 was the first isolated JCVNCCR variant, it has subsequently been shown that many JCVisolates from PML patients do not contain the second TATA boxand that it may be dispensable for viral replication (293, 317). Anaturally occurring variant of the NCCR, found in the CY or“archetype” JCV sequence, is composed of a single copy of the98-bp repeat of a-c-e, with 23-bp (“b”) and 66-bp (“d”) sequenceblocks between “a,” “c,” and “e” to yield an a-b-c-d-e structure(Fig. 3C). Archetype is rarely associated with PML tissue (554).The consistent isolation of tandem repeat-like NCCR sequencesincluding the 98-bp tandem repeat in tissues obtained from PMLpatients strongly suggests the importance of this structure in viralpathogenesis (154, 214, 317, 322, 510). The prototype and proto-type-like sequence variants are generally found in PML tissue,while kidney- and urine-derived NCCR sequences are normallyhomologous to archetype.
Comparison of the prototype NCCR with the archetype NCCRallowed identification of six blocks of sequence, “a” through “f”(24), and illustrated that archetype NCCR contains all of the nu-cleotide elements present in repeat structures as depicted in Fig. 3.Based on this comparison (24) and suggestions from BK virusstudies (144, 427), it has been proposed that all JCV isolates con-tain NCCRs that are derivatives of the archetype sequence (144,205, 554). Despite the presence of functional protein-coding re-gions and an origin of replication, archetypal NCCRs do not sup-port robust growth in culture (96) and are isolated almost exclu-sively from the kidneys and urine (144, 286, 554). A mechanismfor derivation of prototype sequence from archetype and dissem-ination of archetype in the host have yet to be demonstrated, al-though the prevailing model holds that archetype-like sequencesare transmitted from person to person and then undergo deletionsand duplications within the infected host, leading to PML-typeNCCR sequences, which traffic to the brain. This “rearrange-ment” of the NCCR may take place in lymphoid cells like B cells,
since they possess the Rag1 and Rag2 enzymes for immunoglob-ulin gene rearrangements.
Several other variants of interest have been identified. TheMad-4 variant is frequently used as a lab strain. It is identical toMad-1 except that the NCCR contains a 19-bp deletion that elim-inates the second, late-proximal TATA box. Mad-4 has a highincidence of oncogenesis in rodents (381). The coding regions ofthe Mad-4 isolate are identical to those of Mad-1 (317), so differ-ences seen in cell culture models are directly attributable to differ-ences in the NCCR. Additionally, an owl monkey inoculated in-tracranially with a donor tumor cell suspension containing Mad-4developed an astrocytoma that was cultured and spontaneouslysecreted infectious JCV into the culture medium. After serial pas-sage in culture, this virus also showed differences in the sequenceand function of large T antigen (308).
In many PML patients, there is a predominant genotype of theNCCR, which probably coexists with minor subtypes with varia-tions. The NCCR varies between patients, as well. Most of thesevariants contain a repeat, with both deletions and insertions com-pared to archetype and prototype NCCRs. For example, one earlyisolate, Mad-8 (Fig. 3C), is more typical of NCCR variants foundin PML patients than Mad-1. This variant contains a repeat struc-ture similar to that of the prototype, with one large deletion andone insertion, as well as several smaller insertions and basechanges (316, 317). Examples of rearranged NCCRs can be foundin many studies that have sequenced, annotated, and aligned mul-tiple NCCRs from PML patients (413, 485, 552, 557).
Additional support for the role of the repeat NCCR structure inpathogenesis has been the identification of prototype-like se-quences in cells suspected of harboring latent virus, includinglymphocytes isolated from peripheral blood (121, 341, 444, 494)and the bone marrow (201, 322, 484). Interestingly, the B celldevelopment from CD34� progenitor to mature plasma cells ca-pable of secreting IgG requires the expression of genes that sup-port and carry out recombination (467). It may be that low levelsof JCV genome in latently infected lymphoid cells are subject torearrangements, which is supported by the environment in thedeveloping B cell.
Regardless of how the repeat NCCR variants are generated, thisform of JCV is the pathogenic form that has been repeatedly iso-lated from PML patient tissues. Because the prototype NCCR con-tains the repeat structure, it contains significantly more bindingsites for the transcription factors essential to viral gene expression.In particular, the archetype sequence does not contain the Oct-6/tst-1/SCIP sites present on the border between regions “c” and “e”(Fig. 3) (510). Additionally, the lack of neighboring “a” and “c”regions eliminates Spi-B binding sites, which are important forearly viral gene expression (314). The lack of binding sites forbrain-specific transcription factors may be what abrogates theability of archetype virus to cause disease in the brain. The lack ofrepeats of region C in archetype also leads to a reduced number ofNFI binding sites, which allow a family of transcription factors tobind the JCV genome and are essential for fully activating viraltranscription in the brain and cells of the lymphoid system. Theseresults indicate the importance of selective repeated binding sitesfor the cellular transcription factors involved in activating viralgene expression.
Although Mad-1 was the first sequenced NCCR isolated from aPML patient and is referred to as the prototype, it appears to be asomewhat atypical isolate. The tandem repeat sequences serve as a
good reference point, however, so newly isolated JCV sequencesare generally compared to the Mad-1 variant. In order to classifynewly sequenced viral NCCRs, a compass-like classificationscheme that organizes NCCR sequences into four distinct varianttypes was developed (213). Variant type I NCCRs contain no in-serts in the a-c-e organization of the NCCR and can be dividedinto variant type IS (singular a-c-e) and variant type IR (a-c-erepeat with no inserts) (e.g., Mad-1 and Mad-4). Variant type IINCCRs contain inserts into the a-c-e sequence and can be classi-fied as variant type IIS (a-b-c-d-e, or archetype-like) or varianttype IIR (containing inserts and repeats) (e.g., Mad-7 and Mad-8)(Table 1).
Viral subtypes and epidemiology. Prior to the system defininggroups of JCV variants by NCCR architecture, several viral typingsystems were developed. Early systems used restriction fragmentlength polymorphism typing (553), which can classify genotypesinto 3 superclasses, A, B, and C (208), but with the increasing useof DNA sequencing, types were defined by genetic sequence. Aregion of 610 bp covering the 3= end of T antigen, the intergenicregion, and the 3= end of VP1 has been used (1, 25, 205). Morerecently, a definitive coding sequence typing system was devel-oped by coding region polymorphisms of 100 full-length JCV se-quences, using predicted amino acid sequences of all the codingregions to define types. Using this system, 7 JCV types were iden-tified, numbered 1 through 8 (type 5 was found to be a minormember of type 3 [3]), each with multiple subtypes (90). Thistyping system has determined the consensus sequence for all viralproteins and the consensus mutations associated with each sub-type.
The different types of JCV are associated with populations ofvarious descent (90) and have been used to map population move-ments (4, 208, 429, 473) as well as for other, diverse purposes, suchas determining drinking water pollution levels (183) and identi-fying cadavers (206, 207). It has been hypothesized that type 6 isthe original JCV type and that JCV coevolved with human popu-lations. JCV split as humans migrated out of Africa, with one typemoving toward Eurasia and the other type only to Europe (384).Type 1 and type 4 are generally associated with Europeans andEuropean-Americans, while type 2A is found generally in Asiansand Native American populations. Types 3 and 6 are isolated pri-marily from Africans and African-Americans. Types 2D and 7Care found among both Asians and South Asians (91, 550). Types2E, 8A, and 8B are found in Western Pacific populations (550).Interestingly, type 8A is found only in populations of Papua NewGuinea (218). JCV subtype 2B, which is more often found amongAsians and Eurasians, has been associated with increased inci-dence of PML (2, 90), while type 4 has been associated with lowerdisease risk (124) (Table 2).
Most of the studies of viral subtype were performed on JCVDNA from urine specimens from infected yet healthy individuals.
More recently, investigation of changes in viral DNA coding re-gions from brain biopsy specimens and CSF specimens from PMLpatients has been undertaken. Alterations in structural proteinamino acid sequences could lead to enhanced viral entry and thuscontribute to PML or, alternatively, could potentially cause dis-tinct diseases. Evidence points to the association of mutations ofcertain amino acid residues in the region of the sialic acid bindingsites and surface loops of VP1 with PML and that these mutationsoccur within the patient after initial infection with JCV (2, 148,172, 286, 477, 556). In at least one case a frameshift mutation inthe VP1 gene was found to be associated with JC virus granule cellneuronopathy (93). In some animal models, changes in VP1 and Tantigen amino acid sequences led to an increased incidence oftumors (123, 147, 308).
In many studies, most of the population appears to be infected,either transiently or latently, with JCV. Epidemiological study ofJCV depends on detection methods, and these methods have var-ied somewhat from study to study. Ten to 30% of adults excreteJCV in the urine, and PCR detection for viral DNA allows foraccurate and sensitive detection of individuals with actively repli-cating virus. Virus has also been detected by PCR in stool samplesand is prevalent in sewage and rivers worldwide (5, 183, 331, 505,506). This has led to the hypothesis that transmission may occurfrom ingestion of nonsterile water. Transplacental transmission isunlikely (43), but transmission from parent to child can occur (42,555).
Not all people infected with JCV excrete virus in the urine;therefore, other methods of determining infection rates have beenutilized. Seropositivity for JCV was first determined using hemag-glutination inhibition assays, but this has since been replaced byenzyme-linked immunosorbent assays (ELISAs), using recombi-nant VLPs (182, 290). More recently, a two-step anti-JCV anti-body assay has been developed in an attempt to stratify disease riskby antibody presence. In addition to a standard ELISA using Mad-1-derived VLPs, samples are adsorbed with JCV VP1 and retestedby ELISA. The percentage of reduction of ELISA detection indi-cates a seropositive sample, reducing false positives while main-taining specificity (171). In order to identify risk factors for PML,further research into epidemiology and subtypes of JCV, as well ascontinued study of transcriptional control of the virus, is required.
Replication of JCV Genomic DNA
JCV has a limited host replication range. There are two identifiedblocks to replication in nonpermissive cell types: early gene tran-scription and DNA replication. Activated late gene transcription isinitiated only after DNA replication begins, although DNA repli-cation is not required for late gene activity in vitro (52, 229). Anumber of host factors are required and may contribute to JCVearly transcription. In the presence of T antigen isoforms, T anti-
TABLE 1 NCCR variants
Varianttype NCCR structure Tissue association Example
NCCR sequenceblock variationsa Disease association
IS Singular, no inserts GI tract, bone marrow a c e f Colon cancer?IR Repeats, no inserts GI tract, tonsil (S, L), bone marrow Mad-1 a c e a c e f PMLIIS Singular, inserts Lymph node, lung, tonsil (L), liver, urine Archetype a b c d e f Urine (nonpathogenic)IIR Repeats, inserts Lymph node, lung, tonsil (S), liver, spleen, urine Mad-8 a b c �e b c �e f PMLa �, one base insertion; lowercase letter, full sequence block; italic b, partial sequence block. All variants are found in the brain and peripheral blood lymphocytes.
Molecular Biology, Epidemiology, and Pathogenesis of PML
gen, the host DNA polymerase, and a number of other cellularproteins participate in JCV DNA replication.
Nuclear domain 10 (ND10) bodies (also called PML nuclearbodies, after the promyelocytic leukemia protein, which makes upthe scaffold of this structure, or PODS, for PML oncogenic do-mains) are discrete nuclear loci characterized by accumulation ofthe promyelocytic leukemia protein, as well as Daxx, SP100, and anumber of other cellular proteins (359). ND10 bodies are the nu-clear substructures at which JCV DNA replication and capsid as-sembly occur (454). ND10 bodies are thought to regulate a num-ber of cellular processes, as well as participate in the life cycles ofnumerous viruses (139). Like for many DNA viruses, the JCVgenome can be detected at ND10 bodies after infection (452).ND10 bodies show a complex biology with regard to viral genetranscription and DNA replication (452). Numerous studies indi-cate that they play a role in cellular defense against DNA viruses,but in a number of cases, ND10 bodies seem to be positive regu-lators of viral replication (139). Multiple viruses, including polyo-maviruses, disrupt or reorganize the ND10 bodies during thecourse of infection (215). Many viruses, including JCV, localize at,or in the vicinity of, ND10 bodies (139, 222, 452–454). DuringSV40 and BK virus infection, DNA elements have been found tolocalize in the vicinity of ND10 (222), and this localization is re-quired for efficient DNA replication but not for transcriptionalactivation of SV40 (489).
The localization to ND10 bodies may promote JCV DNA rep-lication in a number of ways. A number of transcriptional activat-ing and DNA replication proteins can be found at ND10 bodies,including CBP/p300 and proliferating cell nuclear antigen
(PCNA) (453). Thus, it is likely that JCV uses the ND10 bodies asa scaffold for DNA replication and viral assembly during earlystages of infection, while late in infection, JCV causes the disrup-tion of these replication sites (453) but does not reduce the totalamount of the ND10 scaffold promyelocytic leukemia protein.Interestingly, T antigen, which is absolutely required for viral rep-lication, also localized to the ND10 bodies.
Functional T antigen is required for JCV replication. This wasconfirmed by the observation that JCV containing mutations inthe T antigen-coding region cannot commence a lytic infection.Both the SV40 and BKV T antigens can bind to the JCV origin andinitiate a lytic infection (86). The SV40 T antigen has greater DNAbinding activity and is more efficient in directing replication thanthe JCV T antigen (44, 86, 294). The SV40 and JCV T antigens areable to bind to the origin of replication in two of the three GAGGCsites (152). Sequences on the late side of second T antigen bindingsite have been shown to be extremely important to replication.These sequences adopt an unusual tertiary structure (15; reviewedin reference 301), and this structure may be required for efficient Tantigen-directed viral DNA replication.
The kinetics of JCV replication indicate a slow process. Even insusceptible cells in which T antigen is already present, DNA rep-lication is undetectable for several days (303). DNA replicationcan be detected at 3 to 5 days postinfection in susceptible cell typesand continues for several weeks (142, 233).
In addition to the cell type restriction for JCV growth at the levelof early transcription, the viral life cycle is also restricted by celltype differences in DNA replication. JCV can replicate in immor-talized primate cell lines expressing the SV40 T antigen (185, 303),
TABLE 2 VP1 types and associated ethnic groupsa
TypeVP1 type change(s) fromconsensusb
Predominantly associated ethnicgroup(s)
Identical VP1sequence
Coding sequences required forcomplete type identification
a Adapted from references 90, 172, 413, and 477.b Parentheses indicate that the change is found in less than 50% of the type. Boldface indicates the major amino acid for the subtype at this position.c Slightly higher PML risk.d Slightly lower PML risk.e Most frequently found in PML brain/CSF.f BC loop (sialic acid binding region).g HI loop (sialic acid binding region).
as well as in primate cell lines expressing the HIV transcriptionalactivating protein, tat (369, 370). However, rodent cell lines im-mortalized with JCV T antigen, monkey cells, or nonglial humancells that do not express T antigen cannot sustain efficient viralreplication. JCV replication could occur in the monkey and hu-man immortalized cells in the presence of T antigen, but in therodent cells, even T antigen expression could not stimulate repli-cation. Thus, it appears that transcription is regulated by cell-specific factors, while the restriction of DNA replication is mostlikely regulated by species-specific factors. These species-specificfactors, which may be a component or components of the DNApolymerase (142), allow JCV DNA replication only in primates.
Viral DNA replication proceeds as early viral proteins accumu-late. Large T antigen binds preferentially to site II, located in theviral DNA replication origin closest to first TATA box (see Fig. 3)in the NCCR (47), but it also binds cellular DNA (375). Whenlarge T antigen binds JCV DNA, it promotes the YB-1/Pur�switch to viral late transcription. Large T antigen also interactswith host cell replication machinery to directly initiate replication.
Replication of JCV DNA has not been as well studied as that ofSV40 DNA but is likely to be similar. Like in JCV, the SV40 ge-nome is a closed circular supercoiled DNA molecule. To initiateDNA replication, large T antigen forms a double hexamer and actsas a helicase and complexes with topoisomerase I, DNA polymer-ase �, and replication protein A (RPA) (60, 141, 360). Large Tantigen also contributes to elongation of the DNA chain by itsinteraction with DNA polymerase �, proliferating cell nuclear an-tigen (PCNA), and replication factor C (273, 501, 529). Replica-tion proceeds bidirectionally, similar to theta replication, andleads to two interlinked DNA circles, which are resolved throughthe action of topoisomerases I and II (reference 360 and referencestherein). It has been proposed that linear SV40 genomes can ini-tiate rolling-circle replication, generating concatemers, and thatthis method of replication may explain some of the recombinationseen in viral variants (105, 122, 459).
In order to promote an environment conducive to DNA repli-cation, T antigen binds to cellular proteins and DNA to inducesignals to drive quiescent cells toward S phase (64, 119, 237). LargeT antigen has been demonstrated to exhibit numerous functions,including interaction with, and inhibition of, the retinoblastomaprotein (pRb) (48, 490, 533) and p53 (110). Interaction with p53also prevents apoptosis induced by checkpoint activation whencells aberrantly enter S phase (112). Additionally, large T antigencan promote viral replication in G2-arrested cells by inducingDNA damage response pathways, and this function was related tobinding of cellular DNA (375).
Small t antigen has been less well studied, but has been shown tointeract with the RB family of proteins, as well as protein phos-phatase 2A (PP2A) (45). The interaction of small t antigen withPP2A appears to prevent the dephosphorylation of the late proteinagnoprotein, and this allows for greater viral replication (440).Reduction in levels of small t antigen or PP2A results in reductionof DNA replication (45, 440). Inhibition of the phosphatase activ-ity of PP2A also drives the cell toward S phase, thus promotingviral replication.
Three alternative splice variants of T antigen exist, i.e., T=135,T=136, and T=165, which share their N termini with large T antigen,and disruption of their donor splice site results in greatly reducedviral replication (498). Although much study remains to under-stand their functions, putative interactions with Rb family mem-
bers have been identified, which also appear to drive cells into Sphase (46).
While it is difficult to separate the effects of increased viral earlygene expression from DNA replication, several proteins have beenimplicated in directly increasing viral DNA replication. The NFIfamily of proteins has been shown in cell-free DNA replicationsystems to increase DNA replication of adenovirus type 2 (50, 347)and the replication of SV40 in vivo (348). NFI proteins have alsobeen extensively shown to modulate JCV replication in vivo (14,235, 483). The isoform NFI-A, which is expressed in several non-permissive cell types, has been shown to decrease viral late proteinexpression (409), while NFI-X (NFI-D) increases viral gene ex-pression and is highly expressed in cells permissive to JCV repli-cation (344). The cellular protein Pur� is also likely to participatein viral DNA replication, as it can bind the origin of replicationand has been shown to repress viral replication (71, 157). Addi-tionally, there is evidence that the protein S�bp-2 decreases viralDNA replication, while its smaller variant, GF-1, which encom-passes only some of the helicase motifs of S�bp-2, may increaseviral replication (78).
It is likely that because of the repeated sequence and nonstan-dard secondary structure of the NCCR, recombination, deletions,and insertions occur during viral DNA replication. These recom-binations, insertions, and deletions can explain the large varia-tions of sequences of NCCRs derived from PML patients. Theprototype Mad-1 NCCR contains two identical tandem repeats(two sets of a-c-e sequence blocks) followed by an f sequenceblock. Most PML-derived NCCR sequences contain some versionof these repeats, with deletions and insertions in some cases.Archetype, or CY virus, which is found primarily in the kidneysand urine and often found in healthy subjects, does not containrepeats and generally has a regulatory region consisting of se-quence blocks a-b-c-d-e-f. One hypothesis for viral transmissionand evolution is that archetype-like virus is the circulating formand that deletions (generally of b and d sequence blocks) thenoccur, followed by duplication of remaining sequence. This thenleads to a pathogenic form of the virus able to replicate efficientlyin glial cells (24, 198). There is evidence that the archetype “d”region may be inhibitory to JCV growth in some cells so that itsdeletion allows productive infection of other cells (173).
Alternatively, since archetype virus is rarely found in tissue out-side the kidney, it is possible that prototype-like viruses are trans-mitted and that sequences are deleted or duplicated through basemispairing and single-strand slippage or through various forms ofDNA recombination. The “b” and “d” blocks of sequence can befound in the human genome (L. J. Marshall and E. O. Major,unpublished data). These could be incorporated through DNA“capture.” This hypothesis has been used to explain the generationof host-substituted SV40 variants (reference 459 and referencestherein) and may apply to JCV as well. Recombination of the JCVNCCR may also be explained by its interaction with cells of theimmune system (see below).
Transcription of JCV Genes
The encapsidated JCV genome is closed, supercoiled, and circularand is bound by nucleosomes derived from the four core histonesof the previous host cell, as determined for SV40 (12, 41, 302, 333,518). Once the genome is delivered to the newly infected cell, itacquires the linker histone H1 and resembles cellular chromatin(302). In the nucleus, the JCV genome serves as a template for the
Molecular Biology, Epidemiology, and Pathogenesis of PML
host RNA polymerase II (pol II) transcriptional machinery. Tran-scription of the JCV early genes occurs in the absence of de novoprotein synthesis and utilizes only host proteins. Much, if not themajority, of the cell type specificity of JCV within human cellsoccurs at the transcriptional level. Regulation of transcription isdependent on the sequence of the NCCR, as well as the availabilityof host transcription factors.
Mad-1 NCCR transcription factor binding sites include fourOct-6/tst-1/SCIP binding sites (528), two Pur� binding sites (79),two YB-1 binding sites (79, 232), two LCP-1 binding sites (479),two GF-1 binding sites (78), four NFI binding sites (13, 14, 455,483), and six Spi-B binding sites (314). The Mad-1 NCCR is com-posed of two 98-bp tandem repeats, each containing a TATA box(Fig. 3A). The transcription start sites for early and late transcriptshave been mapped in several cell types and for several viral vari-ants. In addition to specifying cellular permissiveness to JCV in-fection, viral NCCR sequence and host factor availability help todetermine transcriptional start sites.
The 5= termini of early mRNAs at 5 days postinfection of pri-mary human fetal glial cells were mapped to nucleotides 122 to125 (using the numbering system introduced by Frisque et al.[154]) by S1 nuclease analysis, which maps to within the late-proximal TATA box. This contrasts with the case for in vitro-transcribed RNA, which mapped to nucleotides 94 to 97 (231). InJCV-transformed hamster brain cells, the start site of major earlyviral RNA was mapped to nucleotides 5115 to 5124, approxi-mately 25 bp downstream of the early-proximal TATA box, whilea second, minor pair of early mRNA start sites was positionedapproximately 25 bp downstream from the late-proximal pro-moter, by which they were probably positioned (310).
The Mad-4 NCCR has a deletion in the second repeat that elim-inates the late-proximal TATA box and allowed for studies thatdetermined the functional significance of multiple TATA boxes.The early mRNA start site of Mad-4 also mapped to nucleotides5115 to 5124 in transformed hamster cells, but the minor startsites were eliminated, indicating that they were indeed positionedby the late-proximal TATA box (310).
Rodent cells are not permissive for JCV replication and aretherefore transformed by JCV, so the transcriptional start sitesmay be different than in the natural human host. However, similarstart sites were found in the human fetal glial cell line POJ, whichconstitutively expresses a functional JCV T antigen expressed byreplication-defective JCV (309). In primary human fetal glial cells,at 3 to 5 days postinfection, the start sites of Mad-1 were found atnucleotides 5122 and 5082 by primer extension, whereas at 10days postinfection, after DNA replication had begun, the earlymRNA start sites shifted and were found at nucleotides 5012,5037, 5047, downstream from the early-proximal TATA box, andat nucleotide 35, which is between the first and second TATAboxes (233). In another study utilizing both S1 nuclease andprimer extension techniques in primary human fetal glial cells,early mRNA start sites were found for Mad-1 approximately 25 bpdownstream of both TATA boxes, at nucleotides 89 to 92 and 5115to 5125 (95). In that study, as in previous studies, the Mad-4 earlystart site at nucleotides 5115 to 5125 remained, but the site posi-tioned in Mad-1 by the deleted TATA box was eliminated.
These data indicated that both TATA boxes were functional forearly transcription and that they may vary in importance depend-ing on the cell type infected. Although the second TATA box di-rects minor mRNA start site positioning, the major early mRNA
start sites are downstream from the first TATA sequence, and thesecond TATA box may be dispensable. Many of the NCCR vari-ants found in PML patients do not contain the second TATA box(90, 173, 317, 413). Recently, it has been shown that the Spi-Btranscription factor binding sites in the second repeat can com-pensate for the loss of the second TATA box (314). Additionally,NCCR variants lacking repeat sequences show greatly reducedearly transcriptional activity in comparison to both Mad-1 andMad-4 (173). Thus, sequences and transcriptional activator bind-ing sites that are important for early gene transcription are presentthroughout the viral NCCR and contribute to viral early tran-scription, even in the absence of a second TATA box.
In human fetal glial cells, at 17 to 19 days postinfection, severalmajor and minor transcriptional start sites for Mad-1 late mRNAwere found to span a large region of the viral NCCR, coveringapproximately 250 bp between nucleotides 5114 and 242, whichare on either side of the repeats (95, 230). These start sites appearto be positioned not by either TATA box but rather by the se-quence TACCTA, which was approximately 30 nucleotides up-stream from the minor start site at nucleotides 90 to 98, as well asthe major start site at nucleotides 198 to 203 (230). The TACCTAsequence can function as a surrogate TATA box, as has beenshown in SV40 (53, 355).
Host transcription factor availability is the determining factorin both the start sites for early transcription, as well as the quantityof T antigen produced. Although NCCRs containing repeats (suchas Mad-1 or Mad-4) and variants isolated from PML strains havegreater transcriptional activity in PDA cells (173), both the arche-type and various PML isolates show increased transcriptional ac-tivity in glial cells rather than cells of nonglial origin (23, 464).Once T antigen is present, the difference in replication fitnessbetween different variants of JCV becomes much less apparent(23). Therefore, the ability of the virus to transcribe the early sideof its genome is a major determinant of cell type specificity of thevirus and has been the focus of much of the research on viraltranscription.
Unlike other human DNA-containing viruses, such as herpes-viruses, JCV does not bring transcriptional activating proteinsinto a newly infected cell. Thus, early transcription is directedentirely by host cell factors. The JCV NCCR contains binding sitesfor a number of transcription factors and transcriptional repres-sors. As shown in Fig. 3, the proteins NFI-X (344, 410), DDX-1(475, 476), LCP-1 (479), HIF-1� (390), BAG-1 (118), NFAT4(311), NF-�B (405, 435), GF-1 (78), SP1 (190, 191, 242), andSpi-B (314) have all been proposed to bind to certain variants ofthe JCV NCCR and activate early transcription in various celltypes, while NFI-A (409), c-jun (240, 410, 432), c-fos (240), SF2/ASF (439), and C/EBP� (425) have been proposed to repress earlytranscription. Of these, the most well-studied JCV-transcriptionfactor interactions have been with members of the NFI family.
The NFI family of cellular DNA binding proteins is critical toJCV transcription and replication (344, 409, 410). These proteinswere first identified as part of the minimal set of proteins requiredfor in vitro replication of adenovirus DNA (106, 176, 351–353).Three NFI binding sites have been demonstrated in the JCV reg-ulatory region (NCCR) by sequence homology and DNase protec-tion assays (14, 483), and one potential, uninvestigated site exists,as identified by sequence homology. The confirmed sites havebeen alternatively labeled in order from the early side of the ge-nome as NFI-A and NFI-B (285) or from the late side of the ge-
nome as NFI I, II, and III. In order to avoid misunderstanding, thesecond convention is less confusing, as four NFI genes exist,NFI-A, -B, -C, and -X (alternatively called -D) (176). The twoidentical sites in the 98-bp palindromic repeat element (NFI II/III)were shown to be more important for early gene transcriptionthan the unique third element on the late side of the NCCR (NFI I)(260, 455). These sites were also shown to be more important forlate gene expression (259). Additionally, these sites appear to con-tribute to the cell type specificity of JCV, as they are bound bydifferent proteins in different cell types (13, 14, 220, 259, 260, 409,410, 483).
DNase I footprinting experiments demonstrated that varioustissues and cell types contained different factors that bound to theJCV NCCR at dissimilar sites (482). This observation was ex-plained when the four NFI genes were discovered, each with mul-tiple splice variants (176). The dimerization, DNA binding, andDNA replication domains of NFI proteins are found in the Nterminus and are separable from the transcriptional activatingdomains (176). All four NFI genes share homology at the N ter-minus and differ at the C terminus, which is responsible for trans-activation and repression activity (176). NFI proteins can homo-and heterodimerize and compete for identical binding sites. Thecharacter of the NFI dimer may influence transcriptional activity.This could explain why overexpression of NFI-X confers the abil-ity to support increased viral activity in cell types normally non-permissive for JCV (344), while overexpression of NFI-A de-creases the ability of permissive cell types to support JCV infection(409). Additionally, NFI proteins also regulate transcription froma number of genes important in cells of neuronal origin (324).
Members of the activating protein 1 (AP-1) family play an im-portant regulatory role in JC virus transcriptional activation (13,240, 410, 432). NFI binding to and activation of JCV are reducedby the presence of c-jun (13, 410). This appears to be due to theoverlapping AP-1 and NFI binding sites in the JCV NCCR, imply-ing that c-jun physically blocks NFI-induced activation. Interest-ingly, this juxtaposition of AP and NFI binding sites occurs atnumerous genes associated with central nervous system cells, suchas those for glial fibrillary acidic protein (GFAP), a cytoskeletalmarker for astrocytes, and myelin basic protein (MBP), a compo-nent of myelin produced by oligodendrocytes (13). Thus, it islikely that activation of JCV is controlled by access to DNA bind-ing proteins available in cells of the nervous system and that acommon regulatory mechanism of JCV and these genes may exist.Further research will yield a greater understanding of the interplaybetween these DNA binding factors, the JCV regulatory region,and genes important for neuronal and glial differentiation.
Both NFI and AP-1 family members interact with large T anti-gen. NFI has been shown in several studies to increase early andlate gene expression in a T antigen-dependent manner (14, 259,260, 285), as well as to contribute to increased viral replication(344, 409, 410, 465). The AP-1 members c-jun and c-fos have beenshown to physically interact with large T antigen and suppress itsactivation of early genes, as well as viral DNA replication (240).Thus, AP-1 family members and NFI family members appear tomake up an antagonistic switch system for JCV gene expressionand DNA replication.
This system may be similar to a better-characterized switch inthe JCV life cycle. The cellular proteins YB-1 and Pur� interactwith the viral large T antigen to regulate the switch from early tolate gene expression (77, 79, 232, 434, 436, 437). Pur� is a strong
activator of early gene expression and binds the viral lytic controlelement (LCE) (77, 79). As T antigen accumulates, it facilitatesbinding of YB-1 to the LCE, and together YB-1 and T antigenincrease the displacement of Pur� from the viral promoter. YB-1and T antigen stimulate late gene expression (77, 79, 232, 436),and thus Pur�, YB-1, and T antigen work as a genetic switch toshift gene expression from viral early to late genes.
Activated late gene expression requires T antigen and occursconcurrently with DNA replication but, at least in the case ofSV40, does not require DNA replication to proceed (229). Thelarge T antigen ORI binding function is not necessary for activa-tion of late transcription of SV40 (229). Instead, T antigen pro-motes late transcription by interacting with components of thebasal transcription machinery, including TATA binding protein(TBP), TBP-associated factors (TAFs), and transcription factors,including Sp1 (239). T antigen may also function directly as a TAF(92).
A number of DNA binding proteins have been implicated inregulation of viral late transcription. Egr-1 (424), HIF-1� (390),GF-1/S�bp-2 (78), BAG-1 (118), and NFAT4 (311) have beenshown to activate late gene transcription, while C/EBP� (425) andGBP-i (402) appear to function as transcriptional repressors. Sub-units of NF-�B have been shown to increase late gene expression(328, 435) and can increase viral expression in response to tumornecrosis factor alpha (TNF-�) stimulation (539). Subunits ofNF-�B also interact with YB-1 (403), which stimulates the switchfrom early to late gene expression.
Additionally, although the functions of the late viral proteinagnoprotein remain to be elucidated, it has been posited to inter-act with YB-1 and may modulate its activity (438). Agnoproteinmay also interact with large T antigen to reduce T antigen en-hancement of late gene expression (433).
The best-characterized cellular protein known to stimulate virallate gene expression is Tst-1, also referred to as Oct-6 or SCIP.Oct-6/Tst-1/SCIP is a POU domain protein known to function inneuronal development (209, 266, 527), thus potentially contrib-uting to the cell type specificity of JCV transcription. Oct-6/Tst-1/SCIP overexpression increases both early and late gene expres-sion (528). Oct-6/Tst-1/SCIP binds to large and small T antigensin vitro and can synergistically increase both early and late geneexpression in the presence of large and small T antigens (416).
LATENT INFECTION AND INTERACTIONS BETWEEN JCV ANDCELLS OF THE IMMUNE SYSTEM
JC virus displays a complex interaction with cells of the immunesystem. Because of the lack of an animal model coupled with vir-tually undetectable viral levels outside the kidney, the study oflatent sites of infection that are affected by altered immune re-sponses has been difficult. Also, determining the immune re-sponse to JCV infection has been generally confined to those pa-tients with altered immune systems in whom JCV replicates andcauses PML. Additionally, certain cells of the immune system aresusceptible to JCV and play a critical role in the viral life cycle aswell as the pathology of PML.
Initial Infection and Latency
Although JC virus has been known to be the causative agent ofPML since the 1970s, the routes of initial viral transmission andsubsequent dissemination to the brain remain to be fully eluci-dated. Without the provision of exogenous T antigen, JCV was
Molecular Biology, Epidemiology, and Pathogenesis of PML
known to replicate only in human cells of glial origin in culture. Ahematogenous route of infection of the CNS seemed likely afterJCV was discovered to interact with B cells in the brain, periphery,tonsils, and bone marrow and to replicate at low levels in B cells(22, 201, 300, 301, 421, 494).
Several years later, it was shown that JCV could infect tonsillarstromal cells and hematopoietic progenitor cells, as well as pri-mary B cells (341, 345). In addition, evidence was provided thatone of the initial sites of infection could be stromal cells of thetonsils (342). These discoveries led to a model in which primaryinfection most likely occurs in either stromal or immune cells ofthe upper respiratory system, either through respiratory inhala-tion or orally through ingestion of virus-contaminated material,possibly from excreted virus in the urine (37). The virus is thentrafficked to the bone marrow and kidneys by infected lympho-cytes, where it can persist for the life of the host. Upon immuno-suppression or a change in immune cell trafficking, the virus mo-bilizes from the bone marrow and crosses the blood-brain barrier,either alone or in conjunction with a B cell. Once oligodendro-cytes become infected, lytic infection begins (201). It is also pos-sible that virus in the kidney replicates at high copy levels, escapesinto the peripheral circulation, is taken up in lymphoid tissuessuch as the bone marrow, and then undergoes rearrangements ofthe NCCR.
Alternatively, the brain or kidney may serve as a site of latency(104). JCV DNA, but generally not protein, has been found in thebrains of both healthy and immunocompromised patients with-out PML or other neurological disorders (31, 107, 386, 485). Thissuggests that JCV has access to the CNS before disease onset andmay travel to, and nonproductively infect, the brains of some im-munocompetent individuals. It is possible that after initial infec-tion by JCV and viral dissemination, possibly through hematopoi-etic precursors or B cells, the virus reaches glial cells of the brain,where it remains latent. This observation leads to model in whichJCV traffics to the brain and remains latent in the CNS. The viruswould remain latent unless changes in the NCCR, which mayoccur before or during latency in the brain, and available bindingfactors occurred in the presence of immunosuppression. How-ever, if these events occur, then upon a reduction in immunesurveillance and control due to compromise or modulation of theimmune system, JCV may reactivate in situ and cause PML. Thispathway, however, does not address the very low incidence ofPML in allograft recipients who are immunosuppressed for sub-stantial periods for graft protection. In both models of viral la-tency, although the site of latency may differ, similar events mustoccur for progression to PML.
Immune Control of JCV: Humoral and Cellular Responses toInfection
Approximately 60 to 80% of humans produce antibodies againstJCV, indicating that the majority of the population has been ex-posed to the virus (247). These rates vary greatly among popula-tions and age groups (305). Additionally, at any given time, ap-proximately one-fifth of the population sheds JCV in urine (299).Only a very small fraction of these individuals become ill, how-ever, and this occurs only in the presence of underlying changes tothe immune system. Thus, in the majority of cases, JCV infectionis controlled by the healthy immune system.
Several observations indicate that the cellular immune responseplays a vital role in control of the virus. PML is an AIDS-defining
illness, occurring in 3 to 5% of HIV-infected individuals (299).Before the advent of the AIDS pandemic, PML was extremely rare,indicating that a reduction of CD4� T cells due to HIV infectionleads to lack of immune control of JCV. Additionally, non-HIV-associated CD4� T cell reduction due to idiopathic CD4� T lym-phocytopenia has been associated with a number of cases of PML(179, 396). Other studies have implicated an impairment of T cellresponses in the development of PML (526), while a cytotoxic Tlymphocyte (CTL) response has been associated with greater con-trol of JCV and longer PML survival rates (129, 249, 250, 278,321). The use of HAART has reduced the rate of PML in HIV-infected individuals, further indicating an important role for acellular immune response to JCV in control of infection (10, 136).
In contrast, the humoral immune response has not been shownto control JCV infection (249, 526). In fact, the virus seems to haveadapted to replicate and disseminate, possibly through B cells andtheir progenitors. JCV is known to remain intranuclear once as-sembled, which may allow viral escape from immune recognition.
Potential Viral DNA Recombination and Replication in Cellsof the Immune System
In addition to serving as a potential site of viral latency, B cells mayplay an important role in the pathogenesis of PML. Since it hasbeen posited that the viral genome recombines and/or rearrangesduring DNA replication, an attractive model is that these eventsoccur in B cells, which can undergo V(D)J recombination. Thishypothesis is bolstered by the observation that diverse viralNCCRs, including archetype-like and prototype-like NCCRs,have been found in the blood and bone marrow (214, 322, 484).
Recombination that results in prototype-like viral NCCRs isassociated with increased viral activity in glial cells (173). JCVinfection has also been shown to upregulate the DNA damageresponse (101, 102), and high antibody titers to JCV are associatedwith increased chromosomal damage in lymphocytes (269, 340).JCV infection of cells in culture as well as cultured lymphocytesalso results in a high degree of chromosomal damage (356, 357).These damaged chromosomes are similar to those seen in SV40-infected cells and may be the source of some of the sequenceblocks found in PML-associated viral NCCRs, such as in Mad-1.Thus, viral recombination may be explained by chromosomaldamage induced by JCV in cells in which recombination and DNArepair mechanisms are active, as may be the case for SV40 (459).These changes may lead to acquisition of transcription factorbinding sites in the NCCR that are important for pathogenesis. Arecent example was described in patients receiving infliximab,where an archetype-like NCCR contained sequences that led toTATA box-associated Spi-B sites known to be important for viralreplication, while JCV in the urine contained an archetype NCCRsequence (32). Additionally, as B cells mature, different transcrip-tion factors that play a role in increased viral proliferation areupregulated.
As early as the 1990s, it was recognized that there are DNAbinding proteins that are common between B cells and glial cells(300, 421) but that do not exist in T cells (421). At least two factorsshown to be important in JCV transcription and regulation,NFI-X and Spi-B, have been shown to be upregulated in B cells,glial cells, and hematopoietic progenitor cells in which JCV canreplicate (314, 334, 344). Evidence that changes in transcriptionfactors can affect viral transcription as B cells mature is increasing,particularly in light of the observation that natalizumab treatment
upregulates factors involved in B cell differentiation, includingSpi-B (281).
Viral Pathway to the CNS
B cells may also carry JCV across the blood-brain barrier. Evidencefor this is found in the fact that PML was first associated with B celllymphoproliferative disorders (57, 198) and that natalizumabtreatment-associated PML occurs concurrent with mobilizationof lymphocytes from the bone marrow to the periphery (406).Additionally, HIV depletion of lymphocytes in the periphery maylead to mobilization of lymphocytes from the bone marrow to theperiphery, and PML may be “unmasked” after the reconstitutionof the immune system in the periphery by HAART treatment(457).
In addition to mobilization of B cells into the periphery, JCVmust cross the blood-brain barrier to initiate infection of oligo-dendrocytes. B cells can carry JCV to the blood-brain barrier,where it may cross as free virus. JCV may also infect microvascularendothelial cells and thereby cross into the brain (75). Alterna-tively, B cells may carry JCV across the blood-brain barrier. Forinstance, in HIV infection, in which a relatively high percentage ofinfected patients develop PML, macrophage chemoattractantprotein 1 (MCP-1) is upregulated, which increases the permeabil-ity of, as well as lymphocyte migration across, the blood-brainbarrier (530). Infected B cells have been found in the CNS of apatient with PML (300). Additionally, infected B cells can transmitJCV to glial cells in culture (73).
The primary working hypothesis for development of PML isthat at least four events must occur for latent JCV to cause lyticinfection of the oligodendrocytes in the brain: (i) the host immunesystem must be compromised or altered, (ii) the viral NCCR mustacquire changes that increase viral transcription and replication inboth B cells and glial cells, (iii) DNA binding factors that bind torecombined NCCR sequence motifs must be present and/or up-regulated in infected hematopoietic progenitor, B cells, and/orglial cells, and (iv) free virus or virus in B cells must cross theblood-brain barrier and be carried into the brain, where virus ispassed to oligodendrocytes and lytic infection takes place. Oncethe virus is in the brain of the susceptible (immunocompromised)host, PML occurs. These events may occur in the bone marrow, inCD34� lymphocyte precursors or B cells in the periphery, or in thebrain.
IMMUNOMODULATORY THERAPIES AND PML
Several immunomodulatory therapies have been associated withPML (66, 67, 146, 169, 245, 258, 262, 358, 463, 504). These thera-pies are promising for the treatment of a number of autoimmuneconditions and lymphoproliferative disorders. PML has beenidentified as a serious adverse event associated with some of thesetherapies, which led the FDA to require a labeling warning. Theknown mechanism of action of each of these therapies also shedslight on mechanisms of immune control of JCV (for a more de-tailed description of the role of immunomodulatory therapies inthe development of PML, see reference 299 and referencestherein).
Natalizumab is one of the biological therapies that carries ahigh risk of PML. It is a humanized monoclonal antibody for thetreatment of relapsing-remitting forms of multiple sclerosis. Itbinds the �4 chain of the �4/�1 and �7 integrin dimer, alsoknown as very late antigen-4 (VLA-4) (135). VLA-4 mediates cell
migration and infiltration in immune signaling. VLA-4 binds toits ligand, the vascular cell adhesion molecule (VCAM), and par-ticipates in facilitation of extravasation of leukocytes through en-dothelial cells to the sites of inflammation (418). The �4 integrincan also dimerize with the �7 integrin, preventing T cell binding tomucosal addressin cell adhesion molecule 1 (MAdCAM-1) andextravasation into the gastrointestinal mucosa (299). Multiplesclerosis is characterized by chronic leukocyte infiltration into thebrain, and natalizumab blocks this infiltration by preventing ex-travasation through cell adhesion molecule binding. Natalizumabtreatment results in an increase in CD34� progenitor cells in boththe bone marrow and the blood (217). It also increases circulatingpre-B and B cells in the periphery and prevents homing of CD34�
progenitor cells to the bone marrow and of pre-B cells to lymphnode marginal zones (254, 289, 299). Natalizumab treatment alsoresults in an increase of factors involved in B cell differentiation,including Spi-B, in the peripheral blood (281). As Spi-B has beenshown to increase JCV transcription, this may be a mechanism forthe high risk of PML in those treated with natalizumab (314).Recently, it has been shown that Spi-B is increased in CD34� cellsand B cells in natalizumab-treated patients (Marshall and Major,unpublished data). The risk of PML during natalizumab treat-ment rises as treatment progresses, and the true incidence of PMLdue to current immunomodulatory therapies remains to be deter-mined, but it has been estimated to be approximately 3.85 per1,000 patients treated with more than 24 infusions (available forprescribing physicians at https://medinfo.biogenidec.com).
Rituximab is an anti-CD20 humanized monoclonal antibodythat fixes complement. Binding of CD20, expressed on B cells,results in downregulation of the B cell receptor and cytolyticapoptosis of CD20� B cells (412). Administration of rituximabresults in depletion of CD20� B cells in the peripheral blood andCSF (299, 330). Rituximab treatment has been associated withsevere viral infections (9) and JCV-induced PML (299). As withnatalizumab, pre-B and B cells may be mobilized from the bonemarrow and lymph nodes to replace depleted CD20� B cells in theperiphery, and there is an association with higher levels of CD34�
progenitors in the periphery (299).Efalizumab is a humanized monoclonal antibody against
CD11a, a subunit of the leukocyte function-associated antigentype 1 (LFA-1), a T lymphocyte adhesion molecule. LFA-1 bindsintercellular adhesion molecule 1 (ICAM-1), which allows migra-tion of T lymphocytes from circulation into sites of inflammation(271). Efalizumab also downmodulates expression of VLA-4 andresults in T cell hyporesponsiveness (177). Efalizumab was volun-tarily withdrawn from the market because of the occurrence ofPML at an incidence of approximately 1 in 500.
Infliximab is a humanized monoclonal antibody against tumornecrosis factor alpha (TNF-�) (246). Infliximab also inducesapoptosis in TNF-�-producing T cells (507, 508). It has been as-sociated with an increase in infections or reactivation of latentinfections (120). This is probably due to a reduction in cellularimmunity due to the blockage of TNF-� and T cell reduction.
Mycophenolate mofetil is a small-molecule prodrug used toprevent rejection of organ transplants (283). It is metabolized bythe liver to become mycophenolic acid, which blocks B and T cellproliferation by inhibiting IMP dehydrogenase and preventingpurine synthesis (407). It is unclear how mycophenolic acid ad-ministration leads to PML.
It is likely that some of these therapies lead to PML due to a
Molecular Biology, Epidemiology, and Pathogenesis of PML
decrease in immune surveillance. Conversely, several of thesetherapies, notably natalizumab and rituximab, result in a decreaseof mature B cells in the periphery and a subsequent mobilizationof immature B cells from the bone marrow, potentially dissemi-nating latent virus to the brain. Recombination of DNA in B cellsalso offers an attractive model for the changes in the viral NCCRthat are necessary to increase pathogenicity and replicative effi-ciency of the virus in glial cells.
PML AND HIV/AIDS
PML is an AIDS-defining illness and is the cause of death in 3 to5% of AIDS patients (299). This rate of disease is significantlygreater than that in patients with other underlying causes of im-munosuppression. This may be due to several factors, including,but not limited to, duration and extent of immunosuppression,changes in cytokine secretion induced by HIV, viral interactionsin coinfected cells, and increased blood-brain barrier permeabilityallowing for B cells infected by JCV to enter the brain (198).
As discussed above, CD4� T cell number and specificity aremajor determinants of JCV infection of the brain and occurrenceof PML. Additionally, CD8� T cell responses specific to JCV areimportant to control of JCV (126, 250, 278, 279, 544). Duringchronic viral infections, CD4� T cells are required to maintain aCD8� T cell response (326). Dysfunction of B cells and increasedcirculation of B cells, which may favor JCV crossing of the blood-brain barrier, have also been observed during HIV infection (35).Thus, HIV infection seems to promote an immunological statethat favors the onset of PML.
Additionally, studies have shown a potential synergistic role ofHIV and JCV at the molecular level, an effect that is likely a causeof the high rate of PML in HIV-infected individuals. Interestingly,HIV and JCV may share an immune cell site of latency, as bothJCV and HIV have been reported to be present in CD34� bonemarrow progenitor cells and may be reactivated upon differenti-ation to B lymphocytes (68, 198, 341, 345). JCV and HIV can alsoboth infect astrocytes, although the fates of infection differ (87,495, 514), and brain-derived progenitor cells (197, 268, 445). JCVand HIV have the potential to directly interact in multiple celltypes.
The HIV tat protein has been shown to increase transcriptionfrom JCV (82–84, 97, 370, 415, 472, 481), while the JCV agnopro-tein may cause a slight decrease in the replication of HIV (224).Archetype JCV, which normally cannot be efficiently propagatedin cell culture, can replicate in cells expressing HIV tat (369, 370).tat has also been shown to be secreted from infected cells andinternalized by uninfected cells, affecting cellular function (137,138, 203, 491, 531) and thereby abrogating the necessity of JCVand HIV coinfection for the molecular interaction between theseviruses. The uptake of HIV tat has been shown in oligodendro-cytes, which supports the possibility of increased JCV transcrip-tion through the interactions of tat with various activatingproteins (97, 133). The viruses also share requirements for tran-scription factors, including members of the NFI family (220, 344),and both viruses interact with the cellular protein Pur� (77, 156,532). tat interaction with Pur� has been shown to increase lateJCV transcription (252) and replication (98). Interestingly, theinteraction between HIV tat and cellular Pur� has been shown toplay a role in DNA repair (525), which could potentially causeincreased JCV rearrangements in coinfected cells, leading to anincreased chance of JCV NCCR sequences associated with PML.
The transcription factor Sp1 may also play a role in the linkbetween JCV and HIV. In one study, Sp1 sites found in the “b”segment of the archetype NCCR were deleted and TAR-homolo-gous tat binding regions were duplicated in AIDS-associatedPML, while in HIV-negative patients, the Sp1 sites were retained.This may be explained by adaption of the JCV NCCR to containbinding elements for HIV tat instead of the Sp1 binding site formore efficient activation in HIV-infected individuals (338). Con-versely to the upregulation of JCV transcription by HIV, JCV ag-noprotein associates with HIV tat in coinfected astrocytes andrepresses tat-mediated HIV transcription, in part by inhibiting tatinteraction with Sp1 (224).
Additionally, HIV infection increases permeability of theblood-brain barrier (BBB) (23, 206). This may be a function of theviral tat protein, which can activate CCL2, leading to increasedBBB permeability (38, 387, 530), and is associated with an in-creased incidence of PML (318). The increased permeability maycontribute to JCV crossing of the BBB in infected B cells or as freevirus. HIV infection of the brain also causes upregulation of cyto-kines which attract lymphocytes (34), as well as an increase in celladhesion molecules which may facilitate crossing of JCV-infectedcells (367). HIV proteins, such as tat and nef, can cause damage toastrocytes (275, 323), and direct infection of astrocytes by HIVmay lead to neuronal damage (87, 495, 514). This damage maylead to increased inflammation and further infiltration by JCV-infected lymphocytes and may help facilitate onset of PML.
Treatment options for AIDS-associated PML are extremelylimited (see reference 192 and references therein). There is noapproved JCV-specific treatment and hence no treatment forPML. As discussed below, many drugs and biological moleculeshave been investigated for PML treatment, but the results in vivohave not been favorable. The sole treatment for PML in AIDSpatients is highly active antiretroviral therapy (HAART), whichhas been shown to have no direct effect on JCV replication andthus is effective against JCV by limiting HIV replication and thustat production, as well as allowing for increased immune function.Immune reconstitution in AIDS-associated PML has the potentialto greatly increase the life spans of PML patients but carries with itthe potential for severe and life-threatening side effect from im-mune reconstitution inflammatory syndrome (IRIS) (reference219 and references therein). In some cases, PML has been “un-masked” by HAART, in that onset of PML occurred after the be-ginning of HAART (457). These observations suggest that virusmay travel to the CSF following remobilization of lymphocytesduring immune reconstitution.
More study is needed to determine the functional interplaybetween JCV and HIV, but it is clear that there is significant inter-action between the viruses at the molecular, cellular, and immu-nological levels. Both viruses are neurotropic, infect cells of theimmune system, cross the BBB, infect astrocytes, and cause de-struction of oligodendrocytes, leading to a decrease in myelin(408).
CLINICAL ASPECTS OF PML
Risk Factors in the Development of PML
Primary infection with JCV is asymptomatic and occurs in immu-nocompetent individuals early in childhood. Thus, the major riskfactor for developing this infection is immune compromise. Thisincludes patients with cancers that involve the lymphoid system,
such as lymphomas, or patients who are on long-term immuno-suppressive therapy for treatment of cancer or autoimmune dis-eases. Patients who undergo organ transplant are also at risk dueto the need for chemotherapy to prevent organ rejection (325).Some elderly patients may also develop PML, presumably due tocompromise in the cellular immune responses associated with theprocess of aging (164). The recent development of monoclonalantibodies that target different arms of the immune system hasalso been associated with PML. The majority of these cases are dueto the use of natalizumab (anti-�4 integrin), which prevents T celltrafficking into the brain and is used for treatment of multiplesclerosis, and rituximab, which targets B cells (anti-CD20). How-ever, cases have also been reported with alemtuzumab (anti-CD52), infliximab (anti-TNF), efalizumab (anti-CD11a), andibritumomab (anti-CD20) (227). Immune compromise alonemay not be sufficient to cause PML. It is possible that other hostfactors may also play a role. For example, in one study, sequencingof the p53 gene, exon 4, showed heterozygosity (Arg-Pro) atcodon 72 in five of six PML patients (394).
Stratification of patients at risk for PML. Currently, a greatdeal of effort is focused on the stratification of those at risk fordeveloping PML. Several factors that do not appear to be predic-tive of PML risk include viruria and viremia. Detection by quan-titative PCR (qPCR) of JCV DNA in urine occurs at a higher ratein HIV-positive individuals than in the general population (272,327), and while natalizumab treatment is associated with in-creased, but possibly transient, viruria and viremia (80, 422), 20 to40% of the general population also demonstrate periodic excre-tion of JCV in the urine. The presence of JCV in the urine corre-lates with higher anti-JCV antibody levels, but viral copy numberdoes not correlate with anti-JCV antibody levels (171). Based onthese observations, the absence of urinary detection of JCV is nota reliable indicator of PML risk, although the presence of JCVDNA is a risk factor in that it confirms that JCV is present in theindividual. Detectable JCV in the serum has not been correlatedwith progression to PML (515), although the risk associated withJCV detection in specific cellular compartments remains to beelucidated.
In natalizumab-treated patients who are dosed monthly, thegreatest risk factors for development of PML are positive anti-JCVantibody status, previous immunosuppressant use, and treatmentduration of greater than 24 to 36 infusions (225, 466, 515) (currentinformation is at https://medinfo.biogenidec.com). No particularduration or type of prior immunosuppressant use has been iden-tified as increasing the risk of PML. Because no evidence of resid-ual immunosuppression existed when patients were started onnatalizumab (225), persistent immunosuppression may not serveas a useful marker for higher risk of PML in natalizumab therapy.Current data do not allow for complete risk analysis of patientsreceiving greater than 36 infusions of natalizumab, but data will beavailable as more patients are treated for longer periods.
Current evaluation of anti-JCV antibody titers as a risk factor isproblematic. Multiple assays are available, including hemaggluti-nation (HA) inhibition assays (357), ELISAs using purified virus(182), ELISAs using virus-like particles produced in either insector human cells (155, 516, 517), and a two-step assay developed byBiogen-IDEC (171). Additionally, assays currently lack a true pos-itive control (such as humanized antibody against JCV VP1) andinstead use pooled human sera of unknown status to provide a“positive” optical density (OD). These assays generally rely on the
optical density of control sera, which are also of unknown anti-body status, for a negative cutoff value, so no titration of levels orabsolute titers can be determined. Assays also often use one dilu-tion of serum, as opposed to serial dilutions of serum, increasingthe possibility of false negatives. With such differences betweenassays, comparisons between tests and even laboratories perform-ing the same assay are difficult.
Seronegative patients are in the group with lower risk for PML,so a test that determines the presence or absence of anti-JCV an-tibodies is currently of value, as long as it is consistent and has alow false-negative rate. The quantitative anti-JCV antibody titermay be of clinical value when deciding on courses of treatment, inthat antibody titer may be of predictive value for PML risk. It willbe important to standardize assays for anti-JCV antibody titervalues to be useful to clinicians, and efforts are being made in thisarea (391). Physicians must rely on patient history, length of ex-posure to immunosuppressants or immunomodulatory drugs,and anti-JCV antibody status to guide treatment decisions. JCVDNA in the urine also demonstrates the presence of JCV infectionbut is not predictive of PML risk. Several informative and recentreviews of PML risk stratification are available (225, 466). Currentrisk information for natalizumab is available from Biogen-IDEC(https://medinfo.biogenidec.com).
Neuropathology of PML
The central feature of the pathology associated with PML is theinfection of the oligodendrocyte with JCV, which leads to lysis ofthe cell. The infection spreads to surrounding oligodendrocytesand results in focal destruction of myelin. The infected oligoden-drocytes have collections of viral particles in the nuclei, which givethe appearance of inclusion bodies and loss of chromatin struc-ture upon examination by light microscopy. The size of the nu-cleus may also increase by as much as 2- to 3-fold. Morphologi-cally normal oligodendrocytes may also be infected with the virus,as seen by immunohistochemistry or in situ hybridization. Thesecells are usually present in areas where the myelin appears to benormal. The multifocal nature of the lesions suggests a hematog-enous spread of the virus to the brain. To a lesser extent, astrocytesare also infected with JCV. These cells appear large, and the nucleiare irregular and lobulated and appear premitotic but do not be-come neoplastic. These cells have been described using the term“bizarre.” Reactive astrocytes are also present, which is a nonspe-cific finding. Although neurons themselves are rarely productivelyinfected by JCV, demyelination leads to axonal dysfunction, andthe demyelinated axon is susceptible to injury by cellular productsreleased by the glial cells. Axonal injury can result in a retrogradeloss of the neuronal cell body. Loss of neurons is likely permanent.
Invading macrophages are commonly seen in the centers oflesions. They act as scavengers and are often laden with myelindebris in the sites of lesions. Macrophages and microglia are notinfected by JCV. In PML patients with HIV infection, there can bemassive necrotic lesions with infiltration by HIV-infected macro-phages (536).
Lymphocytes are not typically seen in PML lesions except ifthere is restoration of the immune system leading to an immunereconstitution inflammatory syndrome (IRIS). Under these cir-cumstances, CD8� T cells are the predominant cell type and arepresent in perivascular regions at sites distant from areas with JCvirus infection as well as in focal collection in the parenchyma inproximity to JCV-infected cells (511, 544). The presence of cyto-
Molecular Biology, Epidemiology, and Pathogenesis of PML
toxic T cells against JCV is considered to be a good prognosticsign. It is postulated that the rapid restoration of the immunesystem may lead to an expansion of activated T cells, which maycontribute to the massive inflammatory response seen in patientswith IRIS and can result in significant morbidity and mortality.
Clinical Features of PML
Signs and symptoms. Symptoms of PML are usually insidious inonset. The initial symptoms often go unnoticed by patients andfamily and are brought to medical attention only when there issignificant impairment of cognition or motor function. It is notunusual for the initial presentation to be mistaken for a stroke.However, symptoms continue to progress gradually over days toweeks. The differential diagnosis in part depends upon the under-lying condition. In patients with HIV infection, the presence offocal symptoms such as hemiparesis, visual deficits such as loss ofvision on one side, aphasia (i.e., inability to either comprehend orexpress speech), or ataxia help differentiate PML from HIV-asso-ciated neurocognitive disorders (HAND) or HIV encephalitis. Inpatients with multiple sclerosis who develop PML while on treat-ment with natalizumab, the differential diagnosis of PML from arelapse of multiple sclerosis can sometimes be challenging, sincemotor symptoms, ataxia, and visual abnormalities can occur witheither disorder. However, a change in personality or cognitiveabilities, new-onset seizures, or aphasia suggests the likelihood ofPML and warrants further investigation. Since PML is multifocalin nature, the associated clinical manifestations may also repre-sent lesions in more than one region of the brain. Although PMLcan affect any part of the brain, it has a predilection for the poste-rior regions of the brain, including the brain stem, cerebellum,and occipital lobe, and hence the clinical symptoms mirror theseeffects (35).
In recent years the realization that restoration of the immunesystem is the best mode of treatment for PML has led to the use ofaggressive antiretroviral therapy in patients with HIV infectionand rapid withdrawal of immunosuppressive therapy in patientswith autoimmune diseases or organ transplants. This results in aninflux of lymphocytes into the brains of these patients with PML,potentially leading to the clinical syndrome of immune reconsti-tution inflammatory syndrome (IRIS). This is a T cell-mediatedencephalitis, and although the cells are needed to control JCV
replication, paradoxically, the associated massive inflammatoryresponse can result in injury to the surrounding brain tissue andresult in deterioration of the neurological symptoms. Occasion-ally the inflammation can be so severe so as to lead to massiveswelling of the brain, resulting in herniation and death of thepatient (488).
Diagnostic testing. Magnetic resonance imaging (MRI) of thebrain can show characteristic features diagnostic of PML. Theytypically show multiple high-signal-intensity lesions on T2-weighted and FLAIR sequences (Fig. 4). They usually involve theuncinate fibers and have a predilection for the posterior parts ofthe brain. The lesions typically spare the gray matter, since JCVinfects mainly oligodendrocytes and some astrocytes. On T1-weighted images, the lesions may appear hypointense. Since theselesions occur when the patients are immunosuppressed, no en-hancement of the lesions is seen when the contrast agent gadolin-ium is administered. However, patients who develop IRIS havevariable degrees of enhancement. Patients with multiple sclerosistypically develop prominent enhancement with IRIS, since theimmune system is intact when the drug is withdrawn, while pa-tients with HIV infection may show subtle enhancement, since thecell counts in the blood are low and hence fewer cells enter thebrain. Magnetic resonance spectroscopy shows an increase in cho-line and lipids suggestive of gliosis and myelin breakdown; N-acetyl acetate is decreased, suggestive of axonal injury (72).
Although antibody testing in plasma can be used to assess therisk of PML prior to onset or use of immunomodulatory therapy,it is not currently of diagnostic significance after the onset of PML.Instead, the confirmatory test for suspected PML is the demon-stration of the presence of JCV DNA in the cerebrospinal fluid(CSF) or brain. Detection of the virus DNA in the CSF by PCR is ofdiagnostic significance but detection in the blood is not, as viremiais sometimes detectable in the absence of PML, while a percentageof PML patients are not viremic (515). The specificity of quanti-tative PCR (qPCR) can be 100% by targeting unique sequenceswithin the JCV T antigen that are necessary for infection. There isno cross-reaction to the human polyomavirus BK virus (BKV orBKPyV) (428) or in specimens from patients with CNS diseasesother than PML (51). The detection sensitivity of some qPCRassays can be as low as 10 copies/ml (428). Along with clinical
FIG 4 Magnetic resonance imaging of PML. (A and B) T2-weighted images show a progressive and exponential increase in high-signal-intensity lesions over aperiod of 1 month in a patient with HIV infection. Lesions are seen in the frontal lobe, the internal capsule, and the splenium of the corpus callosum with spreadto the opposite hemisphere. (C) A section from the frontal lobe of the same patient shows effacement of the cortical sulci and some midline shift suggestive ofinflammation due IRIS.
evidence and MRI, qPCR results help confirm a diagnostic resultfor PML. Detection of JCV DNA by qPCR and the copy number ofviral DNA can be used for diagnosis in conjunction with clinicaland radiographic findings. Patients with lower, or decreasing, lev-els of JCV genomes in the CSF after therapy may show betterlongevity (108, 109), but the prognostic significance of the viralload of JCV in the CSF has not yet been fully established (51).
In patients with untreated HIV infection, the viral copy num-bers are usually quite high and easily detectable, but in patientswith multiple sclerosis where the immune system is relatively in-tact, the copy numbers of the virus can be quite low and moredifficult to detect (430). In the era of HAART, HIV patients withPML and low or undetectable JCV copy number have been de-scribed with increased frequency (319, 320), and HAART treat-ment is correlated with a reduction in the viral load of JCV in theCSF (108, 166). Brain biopsy is sometimes indicated in patientswhere the CSF cannot be obtained or is inconclusive and the MRIis not characteristic of the disease. In the brain tissue, the infectedcells can be demonstrated by immunohistochemistry, in situ hy-bridization, or PCR analysis (8). Other changes in the CSF arenonspecific, with a mild increase in protein but a normal cellcount and glucose. Interestingly, despite immune reconstitution,some patients may not clear the virus completely, and it may per-sist in the CSF (430). While the significance of this persistent virusis not clear, it indicates that JCV can remain in the brain for longperiods despite reconstitution of the immune system or frankIRIS.
TREATMENT OF PML AND PML-IRIS
The prognosis of PML is generally poor. In the pre-AIDS era andbefore antiretroviral drugs were available, death was nearly uni-versal, with an average survival of 9 months in non-HIV patients(474) and 2 to 4 months in patients with HIV infection (36).Although survival of patients with PML has improved due to theuse of antiretroviral drugs, early recognition, and improvement indiagnostic techniques, the mortality rate is still nearly 50% inHIV-infected patients, and while in the multiple sclerosis popula-tion the mortality is lower, the morbidity is severe in the survivors.
Currently, there is no specific antiviral drug against JC virus.Anecdotal reports of response to various treatments are scatteredthroughout the literature. However, all controlled studies havefailed to show any efficacy of the drugs tested against PML. Thisincludes cidofovir (CDV), cytosine arabinoside (Ara-C), and me-floquine (55). The best treatment for PML is the restoration of theimmune system, although even this is not ideal, since it can lead toIRIS. It is hence recommended that while interventions are beingmade to restore the immune function, such as initiation of anti-retroviral agents in HIV-infected individuals and removal of theoffending chemotherapeutic agent, the patients should be closelymonitored for the development of IRIS and treated with steroidsaccordingly (219).
Many broad-spectrum nucleoside analog chemotherapeuticsthat target DNA replication have been used to inhibit JCV repli-cation in PML patients without much success, including cytosinearabinoside (Ara-C), adenosine arabinoside (Ara-A), azidothymi-dine (AZT), acyclovir (ACV), and cidofovir (CDV) (315). Nucle-oside analogs interrupt RNA and DNA synthesis and therefore canbe used as potent antiviral agents; however, they are also highlytoxic due to interruption of host RNA and DNA synthesis. Ara-Cand, to a lesser extent, AZT have been shown to limit JCV repli-
cation in a tissue culture model (196) but to vary in their ability toinhibit JCV in PML patients in vivo. Ara-C monotherapy (7, 59,109, 159, 274, 313, 364, 376, 393) or in combination with CDV(81, 184, 492, 520), methotrexate (162), or interferon (159, 189,471) has been reported with positive prognosis in some cases anddeath in other cases (18, 89, 180, 195, 204, 346, 404, 462, 502). TheACTG 243 clinical trial showed no benefit in survival rates forPML patients treated with either intrathecal or intravenous ad-ministration of Ara-C. It was unlikely, however, that the drug everreached the multiple plaque lesions in the AIDS/PML patients inthat trial (180).
Cidofovir (CDV), an acyclic nucleotide phosphonate analog ofdeoxycytosine monophosphate effective for treatment of cyto-megalovirus retinitis, has shown antiviral activity for nonhumanpolyomaviruses using in vitro cell cultures (16), but most cases ofCDV use for treatment of PML demonstrate no benefit (109, 312,546). More recently a hexadecyloxypropyl lipid conjugate ofCDV, commercially known as CMX001, was shown to inhibit JCvirus replication in cell cultures derived from human fetal brain(174, 216). In 2010, CMX001 was used in combination with inter-leukin-7 treatment in a patient with PML and idiopathic CD4�
lymphocytopenia, resulting in a significant reduction of viralloads and improvement in clinical symptoms over 8 weeks (382).Importantly, both in vitro studies reported significant levels oftoxicity as measured by decreasing cell viability with increasingconcentrations of CMX001 treatment, and toxicity remains animportant consideration for CMX001.
Another pathway targeted for treatment of PML includes inhi-bition of virus entry into the host cell and presumably of spreadbetween cells, limiting the progressive nature of the disease. JCVenters into host cells through binding of the virus to the primaryreceptor �2,6-linked sialic acid moieties (125, 361) and the sec-ondary receptor serotonin receptor 2A (5HT2AR) (130, 361) onthe cell surface. Because sialic acid moieties are common amongall cell types, including cells not permissive to JCV infection,blocking JCV binding to this molecule is likely not an effectivetarget for therapeutic intervention. More recent studies have fo-cused on blocking JCV binding to 5HT2AR on permissive cells inthe brain using serotonin receptor agonists (385). Blocking accessto 5HT2AR using antibodies or the serotonin receptor agonistschlorpromazine and clozapine was effective in limiting JCV infec-tion in human brain-derived cell cultures (21, 30, 130, 371, 373,442); however, these drugs have serious side effects and toxicityissues. Newer antipsychotics, including zisprasidone, risperidone,and olanzapine, were shown to inhibit JCV infection up to 10-foldmore potently than the previously studied agonists in an in vitrosystem (11). Based on these in vitro results, serotonin receptoragonists have been used to treat PML with various degrees of suc-cess. Treatment of PML with mirtazapine alone (263) or in com-bination with Ara-C (520) was associated with a favorable out-come in some patients, while combined chlorpromazine and CDVtherapy was ineffective in lowering JCV levels in either the CSF orplasma (392). Subsequent studies using human brain microvas-cular endothelial cells (74) and human fetal progenitor-derivedastrocytes and oligodendrocytes (343) showed that JCV infectionof these cells occurs independent of 5HT2AR expression, suggest-ing that 5HT2AR is not sufficient or essential for JCV infection ofcertain cell subsets in the human brain. Further studies are war-ranted to determine the efficacy of serotonin receptor agonists astreatment for PML.
Molecular Biology, Epidemiology, and Pathogenesis of PML
The antimalaria drug mefloquine was shown to inhibit JCVreplication using in vitro cell culture models derived from humanfetal brain in an attempt to identify FDA-approved, commerciallyavailable drugs/biologically active molecules with antiviral activi-ties against JCV (56). Mefloquine is known to cross the BBB andaccumulate in the brain, where JCV infection is pathological (221,388), but has been associated with neurotoxicity (493). Severalindependent case reports showed that mefloquine treatment ofPML was successful in reducing the viral burden in the brain andwas associated with improvement of clinical symptoms (168,244). However, as reported at the 2011 annual meeting of theAmerican Academy of Neurology, a multicenter clinical trial sup-ported by Biogen-IDEC Inc. and Elan Pharmaceuticals failed toshow a reproducible reduction in the JCV DNA in PML patientCSF or reduced clinical progression of PML in response to meflo-quine treatment (150).
Although development of PML no longer guarantees fatality,as described above, the prognosis is poor and therapeutic optionsare few and regularly ineffective. Withdrawal from immunosup-pressants in non-AIDS PML and HAART therapy in AIDS-relatedPML have been associated with immune reconstitution in thebrain and control of viral replication. Rapid immune reconstitu-tion is important to CNS immunosurveillance and control of JCVreplication (33, 129, 161, 223, 249); however, IRIS itself can be aserious, often fatal outcome. Low T cell counts and high numbersof copies of JCV DNA in the CSF at the time of PML diagnosis areclear risk factors for death (66, 128, 160, 249). Early use of five-drug combined antiretroviral therapy after PML prognosis hasbeen shown to improve survival, which is associated with recoveryof anti-JCV T cell responses and reduction of JCV DNA in the CSF(160). Adoptive transfer of JCV antigen-specific cytotoxic T cells,generated after in vitro stimulation with the viral T and VP1 pro-teins, concurrent with citalopram (a serotonin reuptake inhibitor)and CDV treatment resulted in clearance of JCV from the CSF andimprovement of clinical symptoms in a hematopoietic cell trans-plant recipient with PML (27). In addition, bioenergetic parame-ters that reflect T cell immunocompetence, such as intracellularCD4�-ATP concentration, has been shown to inversely correlatewith risk of infections during immunosuppressive therapy, in-cluding infections with the human polyomaviruses BKV (28) andJCV (178). These studies continue to demonstrate the essentialrole that the immune system plays in both development of PMLand control of JCV replication. As a result, the development offuture therapeutics for PML should focus not only on blockingviral replication but also on reconstituting an effective T cell re-sponse against the virus in the brain.
OTHER JCV-ASSOCIATED DISEASES
JC Virus Granule Cell Neuronopathy and Other JCV-Associated Neurological Disorders
While the typical characteristics of PML are those of a white mat-ter disease, productive infection of granule cell neurons by JCVhas been reported. Changes in the cerebellar granule layer duringPML have been observed for over 50 years (419), and these includeenlarged and hyperchromatic nuclei (420), which are found inapproximately 5% of PML patients. For many years it was unclearwhether these cells were directly infected with JCV or were dam-aged collaterally as a result of the destruction of glial cells by JCV.
In 2003, productive infection by JCV of granule cell neurons in
the cerebellum in an HIV-positive patient with PML was de-scribed (127). This individual presented with pyramidal tract andcerebellar dysfunction. Autopsy revealed classic PML in the fron-tal lobe and productively infected neurons expressing viral pro-teins in the internal granule cell layer, as demonstrated by immu-nohistochemistry and electron microscopy. Infected neuronswhich did not express viral proteins were also detected by in situhybridization.
Subsequently, JCV was found in the brain of a patient withcerebellar atrophy but no detectable white matter PML lesions(251). This pathology, termed JC virus granule cell neuronopathy(JCV-GCN), was proposed to be a novel clinical syndrome dis-tinct from PML. JCV-GCN has been since described in both HIV-positive and HIV-negative patients (175, 188, 228, 448, 487). Inthe original case study, it was determined that the NCCR was typeIS, with no “b” and “d” sequence blocks as found in archetype andno repeat structure but with some other small insertions that didnot correspond to sequences found in any known JCV isolate. Amore recent case involving an AIDS patient with cerebellar atro-phy also showed rearrangement of the NCCR, also with a typeIS/IIS arrangement (with no sequence block “b” and only a partialpiece of block “d”). Comparison of CSF-isolated virus and cere-bellar virus NCCRs showed differences and changes in transcrip-tion factor binding sites (426). Thus, it appears that alternativetranscription factor binding sites may play a role in JCV-GCN,further demonstrating the importance of the NCCR sequence inJCV cell type specificity.
Alternatively, entry into neurons or postentry steps such astransport, uncoating, or assembly may be changed by differencesin JCV capsid proteins. In multiple JCV-GCN case reports, dele-tions and frameshift mutations in the C terminus of the JCV VP1gene have been found (93, 94) when matched to the Mad-1 variantas well as to a hemispheric white matter isolate from the index caseof JCV-GCN.
JCV-GCN is a productive infection of granule cell neurons andshould be considered in cases of cerebellar atrophy. Symptoms areindicative of subacute or chronic cerebellar dysfunction, includ-ing gait abnormalities, dysarthria, and incoordination (486). MRIfindings of cerebellar atrophy in the absence of white matter le-sions and positive PCR of the CSF indicate the possibility of JCV-GCN (486). Diagnosis is complicated by the possibility that bothJCV-GCN and classic PML pathologies can occur simultaneously.The finding of JCV-GCN in conjunction with classic PML re-quires cerebellar biopsy showing a lytic infection of granule cellneurons. It is possible that JCV-GCN, or productive infection ofneurons in general, is a frequent complication in classic PMLcases, with a report of 79% of PML cases showing infection ofgranule cell neurons (542). It is also possible that classic PML, withlesions of undetectable size, may be present in the reported casesof JCV-GCN.
JCV protein expression in cortical neurons neighboring whitematter lesions of PML patient samples has also been detected,although the predominant expression was of T antigen, indicatingthe possibility of an abortive or restrictive infection in these cells(545). In one case, termed JCV encephalopathy, lytic infection byJCV of cortical pyramidal neurons with limited demyelinationwas described (543). Another rare pathology of JCV, JCV menin-gitis, has also been reported (40, 512). While these case reports areof interest in that they demonstrate a possible expansion of per-missive cell types and clinical pathology of JCV, this review focuses
on classical PML and PML-IRIS, as this has been the focus of themajority of research on JCV. More detailed information on rareJCV-associated diseases can be found in a recent review article(486).
Potential Association of JCV with Human Cancer
In addition to its role in the development of PML, several studieshave underscored the ability of JCV to transform cells in cultureand induce tumors of neural origin in several experimental animalmodels, such as golden Syrian hamsters and rats (114, 287, 288,374, 523).
Early studies also confirmed that JCV caused tumors in nonhu-man primates, specifically New World monkeys, including owland squirrel monkeys. While several different kinds of tumorsform in JCV-inoculated rodents, JCV-induced tumors in owl andsquirrel monkeys are almost exclusively gliomas. These gliomasdevelop 14 to 36 months after intracranial inoculation with JCV,with a long period of asymptomatic “latent” infection followed byrapid tumor growth and progression, neurological impairment,and death (200, 287, 288, 329). New World monkeys have beenused to study the immune response to JCV-induced tumors (524),as well as to develop radiographic diagnostic procedures for hu-man astrocytomas (199).
Most JCV-induced brain tumors in monkeys have integratedviral DNA (335), often in a head-to-tail configuration of at leasttwo copies (336), and express the large T antigen (298). T antigendid not bind the p53 tumor suppressor protein (304). Interest-ingly, in another case, JCV T antigen was found to bind p53, in-dicating that there were structural differences between T antigenin this tumor and that from the previous study (308). This was alsothe first case of p53 described in a primate brain tumor. In thisstudy, a glioma was induced in an owl monkey inoculated withJCV, and recipient owl monkeys were inoculated intracraniallywith a tumor cell suspension of the explanted glioma tissue. Onemonkey (termed owl monkey 586), developed an astrocytoma,which, upon explant, was shown to have both integrated and freeviral DNA with an NCCR corresponding to the Mad-4 variant.These cells also produced JCV T antigen which bound p53 andproduced infectious virus particles. Studies of gliomas in NewWorld monkeys demonstrated the possibility that JCV couldcause gliomas in nonhuman primates and that, at least in rarecases, these transformed cells could produce infectious virus.These findings provided an impetus to determine whether an as-sociation between JCV and human cancer could be found.
Recent studies point to the association of JCV with human can-cer, including brain tumors, colon cancer, and others, but it isimportant to note that there is a substantive variation betweendifferent laboratories as to the frequency of association of cancerand the presence of JCV (170, 423). It is now evident that theoncogenic activity of JCV is closely linked with the expression ofthe portion of its genome carrying the early gene, whose product,large T antigen, has the capacity to associate with several impor-tant cellular proteins that are essential for the control of cellgrowth and proliferation (117, 534). Direct evidence for tumori-genicity of T antigen comes from studies of transgenic animalsencompassing the JCV early genome, demonstrating that in theabsence of viral replication, expression of T antigen induces abroad range of tumors of neural origin, including tumors of glialorigin, primitive neuroectodermal tumors (PNETs), abdominalneuroblastoma, malignant peripheral nerve sheath tumors
(MPNSTs), and pituitary tumors (114, 145, 256, 257, 456, 460).Histological and molecular studies of tumors from these trans-genic mice revealed the interaction of T antigen with well-charac-terized tumor suppressor proteins, including p53 and members ofthe pRb family, in these tumors and dysregulation of related cellcycle controllers, including cyclins, cdks, and p21WAF (256). Fur-thermore, the association of T antigen with other tumor suppres-sor proteins, including the neurofibromatosis 2 gene product(merlin or NF2), has been observed in JCV transgenic mice withMPNSTs (456).
Extensive study of tumors derived from T antigen transgenicmice have elucidated several other pathways that may contributeto the development of PNETs caused by JCV. In one series ofstudies, it was demonstrated that T antigen, by association with�-catenin, results in the stabilization of �-catenin and its nuclearlocalization in the tumor cells. Interestingly, upregulation of theexpression of several targets for �-catenin was observed in thesecells, including cyclin D and c-myc (158). On the other hand, inthe presence of T antigen, the cross-communication of �-cateninwith G protein kinases, including Rac, in the cell membrane wasfound to activate alternative oncogenic signal transduction path-ways involving JNK, Ras, NF-�B, and others (39).
In addition to �-catenin, JCV T antigen also interacts with in-sulin receptor substrate-1 (IRS-1), a key mediator of the insulin-like growth factor-1 (IGF-1) pathway. This interaction promotesnuclear localization of IRS-1 in mouse medulloblastoma cell linesthat are positive for T antigen (265). While the exact biologicalimportance of this event remains to be determined, recent studiessuggest a role for nuclear IRS in chromosomal instability (414,496). Thus, current evidence indicates that JCV T antigen, by al-tering the biological activities of several tumor suppressor pro-teins, as well as proteins involved in multiple signaling pathways,can initiate a cascade of events that leads to uncontrolled cell pro-liferation in experimental animals containing the JCV genomeand in tumor cells derived from these animals.
Several reports indicate that different forms of human cancer,most notably medulloblastoma, are positive for the presence ofthe JCV genome and the expression of JCV T antigen as well as thelate auxiliary protein agnoprotein, but not viral capsid proteins, inthe tumor tissue (113, 255). These observations remain somewhatcontroversial, as other reports indicate that JCV is not the caus-ative agent of these tumors (186, 241, 349, 509). Interestingly, p53was reported to be colocalized in the nucleus with T antigen, sug-gesting that the interaction of T antigen with p53 may abrogate therole of p53 in control of cell proliferation. In addition, IRS-1 wasfound in the nuclei of these tumor cells, as was also the case fortumors arising in mice that are transgenic for the JCV early region(115). The detection of JCV agnoprotein, which exhibits the abil-ity to dysregulate the cell cycle via p53 and cyclin B and to impairDNA repair through Ku70/80 (100, 101), has also been reported inhuman tumor cells (113). Agnoprotein was reported to be local-ized in the perinuclear region, as is also observed in PML (236).
The JCV genome has also been reported for several tumors ofnonneural origin, including colorectal cancer, gastric cancer,esophageal carcinoma, and lymphoma (65, 69, 111, 116, 131, 132,157, 193, 261, 280, 282, 340, 350, 365, 417, 447, 449, 450, 513, 549),although it should again be noted that there are also reports to thecontrary (163, 291, 363). Consistent with observations in tumorsof neural origin, examination of these tumor cells showed expres-sion of T antigen, and in some cases agnoprotein, in tumor cells,
Molecular Biology, Epidemiology, and Pathogenesis of PML
where it was colocalized with p53 and �-catenin (65, 111, 116, 131,261). While the importance of these observations in regard to theinitiation of transformation or in maintenance of tumors remainsto be established, the detection of JCV viral proteins in these tu-mor cells opens the possibility that JCV T antigen and possiblyagnoprotein may accelerate the development and progression oftumors by inactivating tumor suppressors and/or dysregulatingsignaling pathways.
The evidence outlined above concerning the association of JCVwith human cancer and the parallel molecular findings observedin JCV-induced tumors in experimental animal models support apotential role for JCV in the pathogenesis of human tumors har-boring JCV DNA sequences and expressing JCV oncoproteins.Nevertheless, consensus concerning the extent of involvement ofJCV with human tumors is still lacking, and this is presumably dueto differences in technical issues that exist between different lab-oratories, which have recently been reviewed in detail (117). It ispossible that the expression of T antigen in JCV-positive cells maynot persist during the course of tumor cell maturation and pro-gression. In fact, several studies have shown that T antigen-posi-tive cells may lose expression of T antigen after extensive passagein culture. The loss of T antigen expression appears to have nosignificant impact on the doubling time of the cells. Interestingly,the gene sequences for T antigen remain intact in these cells, sug-gesting that loss of T antigen may result from other mutations inthe JCV genome, modification of the viral DNA and/or RNA, orrapid turnover of T antigen due to its degradation. These obser-vations have led to the speculation that transient expression of Tantigen reprograms pathways that control cell growth and prolif-eration by dysregulating factors involved in the control of the cellcycle and chromosomal stability such as p53. Examination of tu-mors from JCV transgenic mice indicates that expression of Tantigen is observed in only a fraction of tumor cell nuclei, suggest-ing extinction of T antigen expression in a subpopulation of tu-mor cells due to genomic instability or acquired mutations thatpromote uncontrolled cell proliferation (535). Similarly, it is con-ceivable that T antigen may also be downregulated in human tu-mor progression. While possible early events in the genesis oftumors containing the JCV DNA sequence have yet be elucidated,it may be envisioned that JCV involvement in tumorigenesis maybe caused by abortive infection, a rare event, which then leads toclades of cells that, upon T antigen and perhaps agnoprotein ex-pression, enter the cell cycle, progressively grow, and becometransformed. In this respect, T antigen may function by targetingtumor suppressors such as p53, pRb and NF2, and signaling fac-tors, including IRS-1 and �-catenin, as determined in the molec-ular experiments with JCV early transgenic mice and human tu-mors described above. In this way, JCV may initiate the formationof, and perhaps contribute to the progression of, tumors of neuralorigin. Thus, further investigation of the possible role of JCV inhuman brain tumors is warranted, and to fully investigate a rolefor JCV with neural and nonneural tumors, large-scale epidemi-ological studies are necessary.
DISCUSSION
In the years since the advent of the AIDS pandemic, PML hasbecome a growing concern, and the incidence of this disease evenafter the widespread use of HAART remains high. With the in-creasing use of biological immunomodulatory therapies for auto-immune and other inflammatory conditions, the incidence of
PML has increased dramatically. Currently, the PML incidence inHIV-infected patients is approximately 3%. As of 1 February 2012there were 207 reported cases of natalizumab-related PML, withan overall incidence estimated at approximately 1 in 500, althoughthe incidence of PML is roughly 1 patient in 250 after 24 months ofnatalizumab treatment and 1 in 100 after 24 months of treatmentin addition to prior immunosuppressant use (current statisticsfor natalizumab-related PML are available at https://medinfo.biogenidec.com). A registry is being established through the Lab-oratory of Molecular Medicine and Neuroscience, National Insti-tute of Neurological Disorders and Stroke, to increase monitoringand information on the incidence of PML in patients with variousunderlying diseases.
JCV maintains a restricted host range. Within the susceptiblehost, JCV can replicate in a small subset of cell types, which makesthe continued study of JCV both extremely important and verychallenging. Only cells of human origin are susceptible to produc-tive lytic infection by JCV. This has precluded the development ofan appropriate animal model for PML, and studies of pathogenic-ity are dependent on development of cell culture models for JCV.
Viral binding to host receptors may partially account for celltype specificity and disease progression, especially in light of evi-dence that some changes in VP1 structure are associated withprogression to PML or other rare JCV-induced diseases. JCV iswidespread globally, but distinct populations show differences ininfection incidence as well as predominant JCV subtypes. VP1changes can divide JCV into 13 distinct types, with other subtypesdistinguished by changes in the other coding regions. These sub-types have distinct ethnic and geographic distributions and havebeen used for various purposes, from mapping ancient populationmovements to identifying cadavers. Subtypes also show differ-ences in PML risk, as VP1 type 2B is associated with a slightlyhigher PML risk, whereas type 4 shows a slightly lower PML inci-dence. More importantly, changes in VP1 that are not associatedwith distinct subtypes are associated with virus from PML patientsbut rarely with that from healthy people. Known PML-associatedchanges in VP1 cluster in the receptor binding region and likelyinfluence infection to some degree.
Although VP1 changes are potentially associated with PML,much remains to be determined about how these changes influ-ence PML risk. It is clear that variations in the NCCR sequenceleading to unique and/or duplicated transcription factor bindingsites greatly enhance the risk of progression to PML. Host and celltype specificities are controlled primarily at the level of transcrip-tion and replication factors. JCV is able to bind and enter most celltypes studied, but in nonsusceptible cell types, the viral life cycle isblocked at the level of early gene transcription.
Because of differences in host factor availability, the host rangeof JCV is complex. In humans, JCV causes a slow, lytic infection,while in rodents and nonhuman primates, JCV infection leads totumor formation, which is primarily due to the absence of DNAreplication due to differences in host factors in these animal mod-els. This leads to an overexpression of T antigen, where its poten-tial to interfere with cell cycle progression is realized as malignanttransformation.
It is unclear whether human cancer can also be caused by JCVinfection, as there is conflicting evidence on both correlation andcausation of malignant transformation by JCV. The most likelycandidates are glioblastomas, colorectal cancer, and lymphomas,as these have the most evidence in support, but it is clear that
continued study is needed in order to determine what, if any, roleJCV infection plays in human cancer. In addition to the study ofdirect roles of JCV in human cancer, the virus is also a useful toolto dissect mechanisms of transformation as well as host antiviraldefense and the intersection of these mechanisms with tumor sup-pression.
JCV-induced oncogenesis is an important area of study; how-ever, the primary disease caused by JCV is PML. As there is noanimal model for PML, the continued advance of knowledgeabout JCV transcription and replication is dependent on advancesin cell culture techniques able to support JCV replication, as wellas tissue from PML patients and healthy donors. An interestingavenue of investigation is the differences in immune function thatlead to PML. CD4� T cells are important, leading to an antiviralCD8� T cell response. It is unclear whether humoral immunityplays a role in controlling infection, as antiviral antibody titer doesnot correlate with PML risk.
Although over half of the global population is infected withJCV, only a small subset of immunosuppressed individuals de-velop PML, and even in the most severe cases, such as in AIDSpatients, the incidence of PML is less than 5%. This suggests thatnot all underlying causes of immunosuppression are the same.AIDS patients vary both in their level of immunosuppression andin specific immune functions, such as immune signaling, activa-tion, cellular migration, and surveillance. Elucidation of the dif-ferences in immune states between those immunosuppressed pa-tients who develop PML and those who do not will lead to greaterunderstanding of JCV and the immune system, as well as to ther-apies of increased effectiveness. These mechanisms will likely haveto be determined through human studies, as recapitulation of im-mune responses in cell culture does not truly mimic in vivo re-sponses, and there are no animal models suitable for immunolog-ical study of JCV and PML.
The discovery that JCV can infect cells of the immune systemprovided insight into the route and mechanism of infection andthe migration of JCV to the brain. It has been proposed that JCVinfects either circulating B cells or stromal cells in the tonsil, byinhalation or ingestion, early in life. Virus may then be dissemi-nated to the bone marrow, kidneys, and/or brain, where JCV canremain latent for the life of the host. JCV has been found to infectCD34� hematopoietic progenitor cells and primary B cells (341).It is probable that CD34� hematopoietic progenitor cells functionas a viral reservoir. Many viruses infect cells of the immune sys-tem, including human cytomegalovirus (hCMV). InterestinglyhCMV also uses CD34� cells as a site of viral latency (295, 332,458, 519) and can transactivate JCV (538). Both viruses can bemodulated by HIV (84, 270, 481), which has also been proposed toinfect and establish latent reservoirs in CD34� cells (68). All threeviruses are also activated by factors involved in B cell differentia-tion or by the differentiation process itself (68, 270, 314). Theinterplay of JCV and other viruses that infect the immune systemand brain is an area that requires further study, especially in thatcoinfection of certain viruses may be a risk factor for the develop-ment of PML.
Additionally, results supporting infection of CD34� hemato-poietic progenitors gives rise to the possibility that mobilization ofthese cells and differentiation in the periphery are a risk factor forPML. Natalizumab treatment for MS, which increases risk ofPML, mobilizes CD34� progenitors from the bone marrow to theperiphery (49, 217, 558), and these mobilized cells show greater
migration toward chemokine stimuli (217). HIV infection, as wellas MS, for which natalizumab is a treatment, also lead to CNSdamage and inflammation and thus to increased chemokine stim-uli (194, 243, 530, 547), which may lead to greater migration ofvirally infected cells to the brain. Thus, it appears that changes inimmune trafficking and maturation may play a large role in thedevelopment of PML.
These immune changes may also influence the rearrangementsin the JCV NCCR required for development of PML. Archetype-like virus has been found rarely in PML brain, although it is almostthe exclusive type of JCV excreted in the urine. The prevailinghypothesis is that the archetype NCCR is the form transmittedfrom person to person. In this model, the virus spreads throughthe body and remains latent in the kidney and bone marrow. Fol-lowing deletions and rearrangements of NCCR sequence, whichchange, delete, and duplicate host transcription factor bindingsites, JCV becomes fit to replicate in glial cells and thus cause PML.It is unclear in which compartment these changes occur, althoughboth archetype and prototype-like sequences have been found inthe bone marrow, making it a likely site of viral DNA rearrange-ments. Additionally, maturation of CD34� cells to mature B cellsleads to an upregulation in DNA recombination factors requiredfor V(D)J recombination, making these cells an attractive site forthe possibility of viral DNA recombination. Mobilization to theperiphery and subsequent maturation of JCV-infected CD34�
cells could lead to these rearrangements, and thus an increasedrisk of PML, in immunosuppressed patients and those on immu-nomodulatory therapies such as natalizumab.
It is important to note that JCV may be found in several sites oflatency. Infection of cells of the brain may occur at different timesthroughout the life of the host. PML may be caused by reactivationof latent virus in the brain due to decreased immune surveillance,as well as trafficking of new virus to the brain due to increasedlymphocyte mobilization. Because of these factors, as well as in-creased viral replication, there may also be a number of variants ofthe NCCR found in the brain during PML. Next-generation se-quencing and additional research may lead to further elucidationof the pathways of viral dissemination.
If the model in which JCV is latent in the bone marrow andmobilizes to the CNS upon changes to the immune system is cor-rect, then progression from latent JCV infection to active PMLlikely requires four changes: (i) a decrease or change in immunefunction, (ii) rearrangement of the regulatory region to increasereplication fitness, (iii) mobilization of infected cells from sites oflatency to the brain, and (iv) upregulation of factors that increaseJCV replication. This is illustrated in the case of Spi-B. Natali-zumab decreases immune surveillance to the brain and, addition-ally, causes upregulation of Spi-B, a transcription factor involvedin B cell development which is also expressed by glial cells, in theperipheral blood (281). TATA box-associated Spi-B binding sitesin prototype-like viral variants are important for viral transcrip-tion (314). Thus, the combination of underlying disease, immunemodulation and changes in trafficking, and upregulation of fac-tors that result in increased viral transcription, in addition tochanges in viral NCCR sequence, lead to an increased risk of PML.Investigation of a number of other cell type-specific factors, in-cluding NFI-X, that have been implicated in viral pathogenesis isrequired to come to a greater understanding of the risk factorsfor PML.
These risk factors are an increasingly important area of study in
Molecular Biology, Epidemiology, and Pathogenesis of PML
the viral pathogenesis of JCV. These factors may eventually beused as markers for PML risk and disease progression and mayinform treatment of PML, as well as underlying conditions with arisk of PML. Stratifying patient risk is an important considerationbeing investigated in the case of immunomodulatory therapies,with the goal of finding alternative treatments for those at highestrisk of developing PML while maintaining the use of these prom-ising new therapies for those at lower risk of development of PML.Host factors may also be potential targets for new PML therapies.
Currently, there is no effective therapy for PML. In the case ofAIDS, immune reconstitution is the only available attempt at“treatment,” and this course is often not available after severeimmunosuppression. Even when immune reconstitution is possi-ble, IRIS is a major risk and can lead to permanent damage ordeath. When PML occurs during immune therapies such asnatalizumab treatment, plasmapheresis is the only course of ac-tion. This may also lead to IRIS, as well as increased symptoms ofthe underlying disease. Several investigational drugs for PML ex-ist, including CMX001. CMX001 has been shown to inhibit JCVreplication in cell culture (216), but it has toxicity and is approvedonly under review by the FDA for cause. Therapies with greaterefficacy and specificity and with lower toxicity are urgently neededfor the treatment of PML. Because of the steadily rising incidenceand significant morbidity and mortality of PML, further investi-gation of the epidemiology, pathogenesis, and molecular biologyof JCV, as well as potential therapies for PML, is of increasingimportance.
ACKNOWLEDGMENTS
W.J.A. is supported by NIH grants P01NS065719, R01NS043097,R01CA071878, and F32NS070687 (CN). K.K. is supported in part by NIHgrants R01NS35000 and R01MH086358. M.W.F. is supported in part bythe Intramural AIDS Research Fellowship from the NIH Office of AIDSResearch. The Laboratory of Molecular Medicine and Neuroscience, aswell as the Section of Infections of the Nervous System, is supported by theDivision of Intramural Research of the NINDS.
We thank Wendy Virgadamo at Brown University for help in thepreparation of Fig. 2 and Patrick Lane of ScEYEnce Studios for art en-hancement. We also thank members of the LMMN for input and criticalreading of the manuscript.
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Michael W. Ferenczy is a postdoctoral researchfellow in the Laboratory of Molecular Medicineand Neuroscience at the National Institute ofNeurological Disorders and Stroke at the Na-tional Institutes of Health (NIH). He obtainedhis Ph.D. in molecular virology and microbiol-ogy in the laboratory of Dr. Neal DeLuca at theUniversity of Pittsburgh School of Medicine,where he studied the role of the transactivatingprotein ICP0 in epigenetic control of herpessimplex virus 1 transcription. His current re-search interests involve the role of the NFI family of proteins in the JCV lifecycle, as well as JCV-HIV interactions that lead to PML.
Leslie J. Marshall is a postdoctoral research fel-low in the Laboratory of Molecular Medicineand Neuroscience at the National Institute ofNeurological Disorders and Stroke at the Na-tional Institutes of Health (NIH). She obtainedher Ph.D. in microbiology and immunology inthe laboratory of Dr. David Ornelles at WakeForest University, where she studied the molec-ular regulation of adenovirus latency in T lym-phocytes involving the leukemia-associatedprotein RUNX1. Her current work as a researchfellow in the laboratory of Eugene Major at NIH led to the novel observationthat the B cell transcription factor Spi-B is expressed in human glial cells andis involved in the molecular regulation of JC virus gene expression in thesecells. Her studies continue to support an important role for JC virus-infectedlymphoid cells in the molecular pathogenesis of progressive multifocalleukoencephalopathy.
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Molecular Biology, Epidemiology, and Pathogenesis of PML
Christian D. Nelson is a postdoctoral researchfellow in the laboratory of Professor Walter At-wood in the department of Microbiology, CellBiology, and Biochemistry at Brown University.He obtained his Ph.D. in comparative biomed-ical sciences in the laboratory of Professor ColinParrish at Cornell University, where he studiedantibody neutralization and the in vitro stabilityand uncoating of canine parvovirus. His cur-rent research interests involve mapping the in-fectious entry pathway of JC polyomavirus anddeveloping novel antiviral compounds that inhibit JC polyomavirusreplication.
Walter J. Atwood is Professor and Vice Chair ofthe Department of Molecular Biology, Cell Bi-ology, and Biochemistry at Brown University.His laboratory focuses on invasion of host cellsby human polyomaviruses. His early work as agraduate student with Len Norking at the Uni-versity of Massachusetts at Amherst led to theidentification of the cellular receptor for SV40.As a postdoctoral fellow with Eugene Major atthe National Institutes of Health, Dr. Atwoodbegan work on the human polyomavirus JCV,focusing on transcriptional regulation of the virus in glial cells and B cells.Since joining the faculty of Brown University in 1995, Dr. Atwood has fo-cused on understanding how human polyomaviruses engage host cell recep-tors to establish infection. His work has led to several major discoveries,including the characterization of the sialic acid-dependent infectious mech-anisms for both JCV and BKV, characterization of the modes of virus entryinto cells for both JCV and BKV, and identification of a JCV receptorcomplex.
Avindra Nath received his M.D. degree fromChristian Medical College in India in 1981 andcompleted a residency in neurology at the Uni-versity of Texas Health Science Center in Hous-ton, followed by a fellowship in multiple sclero-sis and neurovirology at the same institutionand then a fellowship in neuro-AIDS at NINDS.He held faculty positions at the University ofManitoba (1990 to 1997) and the University ofKentucky (1997 to 2002). In 2002, he joinedJohns Hopkins University as Professor of Neu-rology and Director of the Division of Neuroimmunology and NeurologicalInfections. He joined NIH in 2011 as the Clinical Director of NINDS, theDirector of the Translational Neuroscience Center, and Chief of the Sectionof Infections of the Nervous System. His research focuses on understandingthe pathophysiology of retroviral infections of the nervous system and thedevelopment of new diagnostic and therapeutic approaches for thesediseases.
Kamel Khalili is a Professor in the Departmentof Biology, as well as the Department of Micro-biology, at Temple University and holds an ad-junct professorship at the University of Milan inMilan, Italy. He received his Ph.D. in microbi-ology from the University of PennsylvaniaSchool of Medicine, where he studied regula-tion of human actin genes in adenovirus-in-fected cells. Following postdoctoral work at theWistar Institute and the National Cancer Insti-tute, he joined the faculty at the Jefferson Med-ical College of Thomas Jefferson University. Later, he became the Founderand Director of the Center for Neurovirology and Neurooncology at MCPHahnemann University School of Medicine in Philadelphia, PA. Dr. Khaliliis a Professor and the Founding Chair of the Department of Neuroscienceand the Director and Founder of the Center for Neurovirology at the TempleUniversity School of Medicine in Philadelphia, PA. His research programfocuses on the viral oncology and the molecular biology of neurotropic vi-ruses, with the goal of understanding the molecular basis of virus-induced orassociated neurodegenerative diseases and cancers of the central nervoussystem.
Eugene O. Major received the Ph.D. degreefrom the University of Illinois Medical Centerin infectious diseases and microbiology and wasa part of a team that established a research cen-ter there on the genetics of viruses that causecancer. Following academic appointments asAssociate Professor at the University of IllinoisMedical School and the Loyola University Med-ical School in Chicago, where he was also Asso-ciate Dean of Graduate Programs, Dr. Majorjoined the National Institute of NeurologicalDisorders and Stroke (NINDS) at the National Institutes of Health (NIH) in1981. He has developed a world-recognized translational research laboratoryin the Intramural Program focusing on mechanisms of viral pathogenesis inthe human nervous system, which includes JC virus-induced demyelination,progressive multifocal leukoencephalopathy, and HIV-1/AIDS-associatedencephalopathy. Dr. Major’s research program has also established a novelhuman brain-derived progenitor cell population whose lineage can be di-rected to all major cell types of the brain, similar to embryonic stem cells.
