Extensive Diversification of Human Immunodeficiency VirusType 1 Subtype B Strains during Dual Infection of a
Chimpanzee That Progressed to AIDSQING WEI AND PATRICIA N. FULTZ*
Department of Microbiology, University of Alabama School of Medicine, Birmingham, Alabama 35294
Received 22 August 1997/Accepted 24 December 1997
A chimpanzee (C-499) infected for more than 9 years with two subtype B isolates of human immunodeficiencyvirus type 1 (HIV-1), one (HIV-1SF2) that replicates poorly and one (HIV-1LAV-1b) that replicates efficiently inchimpanzees, died of AIDS 11 years after initial infection (F. J. Novembre et al., J. Virol. 71:4086–4091, 1997).Nucleotide sequence and phylogenetic analyses of the C2 to V5 region of env (C2-V5env) in proviral DNA fromperipheral blood lymphocytes obtained 22 months before death revealed two distinct virus populations. One ofthese populations appeared to be a recombinant in env, having the V3 loop from HIV-1SF2 and the V4-V5 regionfrom HIV-1LAV-1b; the other population had evolved from HIV-1LAV-1b. In addition to C2-V5env, the entire p17gag
and nef genes were sequenced; however, based on nucleotide sequences and phlyogeny, whether the progenitorof the p17gag and nef genes was SF2 or LAV-1b could not be determined. Compared to the two original viruses,the divergence of all clones of C2-V5env ranged from 9.37 to 20.2%, that of p17gag ranged from 3.11 to 9.29%,and that of nef ranged from 4.02 to 7.9%. In contrast, compared to the maximum variation of 20.2% in C2-V5env
for C-499, the maximum diversities in C2-V5env in proviruses from two chimpanzees infected with HIV-1LAV-1bfor 9 and 10 years were 9.65 and 2.48%, respectively. These results demonstrate that (i) two distinct HIV-1populations can coexist and undergo extensive diversification in chimpanzees with progressive HIV-1-induceddisease and (ii) recombination between two subtype B strains occurred even though the second strain wasinoculated 15 months after the first one. Furthermore, evaluation of env genes from three chimpanzees infectedwith the same strain suggests that the magnitude of HIV-1 diversification could be related to higher viralburdens, manifestations of disease, and/or dual infection.
The only nonhuman primate species that can be reliablyinfected with multiple isolates of human immunodeficiencyvirus type 1 (HIV-1) is the chimpanzee (Pan troglodytes). Com-pared to primary HIV-1 infection in humans, the virus can betransmitted to chimpanzees by the same routes, viral burdensin peripheral blood and lymph nodes peak during the first 4 to8 weeks, and detection of HIV-1-specific immune responsescoincides with partial clearance of free virus from plasma anddecreased numbers of infected cells in blood and lymph nodes(1, 18, 21). Although chimpanzees have become infected afterexposure to multiple clade (subtype) B isolates as well as iso-lates from other clades (3, 16, 21, 52, 64), in general, long-termpersistent infections can be established only with HIV-1 strainsclassified as dualtropic and syncytium inducing (SI) (3, 17, 64).This limitation to productive infection with HIV-1 SI strains invivo is also true in vitro (17, 63, 64). The only exception ap-pears to be a non-SI strain that was isolated from a naturallyinfected chimpanzee (57). Despite the association in humansof HIV-1 strains having the SI phenotype with loss of CD41
cells and progression to AIDS (62), reports of HIV-1-relateddisease in infected chimpanzees have been rare (1, 23, 39, 51).Because of this failure to develop AIDS by a subset of morethan 150 chimpanzees that have been infected with HIV-1 forup to 13 years, many investigators feel that HIV-1 infection ofchimpanzees is not a good model to use for evaluating candi-date vaccines. However, immunization with HIV-1 antigensand challenge of chimpanzees, with either cell-free or cell-
associated, homologous or heterologous virus, have providedvaluable information about the feasibility of certain vaccineapproaches and their protective efficacy (4–6, 22, 28–31).
Some possible reasons for lack of HIV-1-induced disease inchimpanzees include innate resistance to the pathogenic effectsof HIV-1; lack of cytopathic effects of HIV-1 on lymphocytes;high levels of CD81 lymphocytes (normal CD4/CD8 ratio ofapproximately 1.0) that mediate suppression of HIV-1 replica-tion (8, 38); no virus-mediated aberrant effects on lymphocytes,such as induction of anergy and apoptosis (15, 32–34, 63);limited exposure to other pathogens due to isolation in restrict-ed-access housing; and infection with HIV-1 strains havinglimited pathogenicity for chimpanzees. However, there is nowevidence that most of these reasons are not valid, and only thelast two possibilities—limited exposure to other pathogens andinherent properties of the HIV-1 strains with which chimpan-zees have been inoculated—are likely to be relevant. The re-cent report of a chimpanzee (C-499) euthanized because ofHIV-1-related severe immunodeficiency and AIDS, as definedby the Centers for Disease Control and Prevention, demon-strated that this species can develop AIDS (54). Furthermore,virus transfused in blood from C-499 to a naive chimpanzeeinduced a rapid decline in numbers of CD41 lymphocytes,indicating that this strain, called HIV-1JC, was pathogenic forchimpanzees (54).
Chimpanzee C-499, which succumbed to AIDS, had beeninfected with the HIV-1SF2 strain for 15 months when it wasexposed intravenously to a high dose of and became infectedwith HIV-1LAV-1b (Fig. 1) (24). This dually infected chimpan-zee was inoculated 6 months later with a third HIV-1 strain,NDK, now known to belong to subtype D (53); however, noevidence that the third strain actually established infection was
* Corresponding author. Mailing address: Department of Microbi-ology, University of Alabama School of Medicine, 845 19th St. South,BBRB 511, Birmingham, AL 35294. Phone: (205) 934-0790. Fax: (205)975-6788.
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obtained. (This early study was performed before PCR wasused routinely for genetic analysis.) Approximately 1 year afterexposure to HIV-1NDK, chimpanzee C-499 was immune stim-ulated by intramuscular inoculation of purified p53gag formu-lated in adjuvant (20). This inoculation coincided with devel-opment of severe lymphopenia, characterized by a significantdecline in CD41 peripheral blood lymphocytes (to a nadir of134 cells per ml of blood), loss of lymphocyte proliferativeresponses to mitogens, and thrombocytopenia; all of theseconditions persisted for more than 1 year and then graduallyreturned to normal levels (23). When C-499 was euthanized, ithad been infected with HIV-1SF2 for almost 11 years and withHIV-1LAV-1b for about 9.5 years. We report here the geneticcharacterization of the HIV-1 quasispecies in peripheral bloodmononuclear cells (PBMC) obtained from this dually infectedchimpanzee 22 months before its death, provide evidence forrecombinant genomes, and compare its quasispecies with thosepresent in two chimpanzees infected only with HIV-1LAV-1b fora comparable time.
MATERIALS AND METHODS
Chimpanzees and virus strains. Three chimpanzees that had been infectedwith HIV-1 for either 9 or 10 years were evaluated; two of these animals (C-459and C-499) were housed at the Yerkes Regional Primate Research Center, andthe other one (C-487) was housed at the Laboratory for Experimental Medicineand Surgery in Primates (LEMSIP), New York University. C-459 had beeninoculated with the first HIV-1 isolate, originally designated LAV-1 and laterLAI (68), before it was passaged in T-cell lines (21). Approximately 5 monthsafter inoculation, whole blood from C-459 was transfused into chimpanzeeC-463, and 2 weeks later, when virus was isolated from C-463’s PBMC bycoculture with normal human PBMC, a large stock of virus was cryopreservedand designated LAV-1b; this virus stock, which was passaged only in primaryPBMC, was used to inoculate both C-499 and C-487 in separate studies (19, 21).At the time C-499 was inoculated with HIV-1LAV-1b, it had been infected withHIV-1SF2 for 15 months (Fig. 1); however, the secondary infection was notprevented, and C-499 became persistently coinfected with two HIV-1 strains(24). Housing and care of chimpanzees were provided according to institutionalguidelines and standard practices for the humane care and use of chimpanzeesin biomedical research. Animals were anesthetized with ketamine hydrochoridefor all blood collections. (Note that chimpanzee C-487, housed at LEMSIP, wasidentified previously in reference 19; this animal is not the same C-487 housed atYerkes and described in reference 21.)
Virus cultures. To isolate virus from chimpanzee PBMC, approximately 107
cells were cocultured with normal human PBMC that had been stimulated for 3days with phytohemagglutinin (PHA)-P as described previously (21). Virus rep-lication was monitored by the production of reverse transcriptase activity incell-free culture supernatants. To determine the least number of CD41 lympho-cytes required to isolate virus from PBMC, CD81 cells were removed by usingDynabeads coated with antibodies to CD8. Aliquots of 10-fold serial dilutions ofthe CD41-enriched cells were added in replicates of six to phytohemagglutinin-stimulated human PBMC in 24-well plates and monitored as described above.The genetic analysis of proviruses in cultured PBMC was done with DNAextracted after 4 weeks of culture.
Genetic analysis. Proviral DNA was amplified by nested PCR in three regionsof the HIV-1 genome: a fragment spanning the C2 to V5 regions of the env gene(C2-V5env), and the entire p17gag and nef genes. Genomic DNA from culturedand uncultured chimpanzee PBMC was extracted with a QIAamp blood kit(Qiagen, Chatsworth, Calif.) and used as templates for nested PCR in a PTC-100Programmable Thermal Cycler (MJ Research). First-round amplifications weredone with 0.25 to 1 mg of genomic DNA for 25 cycles at 94°C for 1 min, 55°C for1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min. A 5-ml (or1/10 volume) aliquot of the first-round PCR products was subjected to a secondround of PCR for 35 cycles. The positions of all primers are given relative tobases in the HIV-1HXB2 molecular clone in the HIV-1 database (53). The outer
(p17/1 and p17/2) and inner (p17/3 and p17/4) primer pairs used to amplify 443bp encoding p17gag were as follows: p17/1 (bases 681 to 704), 59-TCTCGACGCAGGACTCGGCTTGCT-39); p17/2 (bases 1242 to 1219), 59-AAGTTCTAGGTGATATGGCCTGAT-39; p17/3 (bases 757 to 780), 59-AATTTTGACTAGCGGATCCTAGAA-39); and p17/4 (bases 1199 to 1177), 59-GTTCTGCAGTATAGGGTAATTTT-39). The inner (D and F) and outer (C and H) primer pairsused to amplify a 681-bp fragment spanning C2-V5env were reported previously(25). For the entire nef gene, a 769-bp fragment was amplified with the outer(nef1 and nef2) and inner (nef3 and nef4) primer pairs: nef1 (bases 8682 to8707), 59-TAGCAGTAGCTGAGGGGACAGATAGG-39; nef2 (bases 9557 to9532), 59-TGGTCTAACCAGAGAGACCCAGTACA-39; nef3 (bases 8749 to8775), 59-ATACCTAGAAGAATAAGACAGGGCTT-39; and nef4 (bases 9518to 9496), 59-TGCTTATATGCAGGATCTGAGGG-39. The resultant PCRproducts were separated on 1% agarose gels stained with ethidium bromide, andthe bands were excised; the DNA fragments were purified by using a Qiagen gelextraction kit and then cloned into the pCRII vector (75), using conditionsspecified by the manufacturer (Invitrogen, San Diego, Calif.). After transforma-tion into competent Escherichia coli cells, plasmid DNA from independent bac-terial colonies was prepared by using a Qiaprep 8 miniprep kit (Qiagen) and thenwas digested with EcoRI to check the inserts.
To estimate the minimum number of proviruses in 1 mg of genomic DNA(equivalent to DNA from approximately 1.5 3 105 cells), 1 mg of purified DNAwas serially diluted 1:2 and added to DNA from a normal chimpanzee to give atotal of 1 mg of DNA. At least two independent, nested PCRs were performedwith each DNA mixture, and the last dilution to yield at least one positivereaction was considered to contain a minimum of one provirus. The products ofeach dilution were analyzed for heterogeneity by heteroduplex mobility assay(HMA), and we also considered the number of unique banding patterns presentfor each dilution of DNA when estimating the minimum number of provirusespresent in a sample. For example, if a 1:32, but not a 1:64, dilution of 1 mg ofDNA was PCR positive, and two distinct HMA patterns were seen, then the 1 mgor 1.5 3 105 cellular genomic equivalents was considered to contain a minimumof 64 proviruses, or approximately one provirus per 2,344 cells.
To minimize the possibility of contamination during PCR, positive-displace-ment pipettors with filtered tips were used, and reactions were performed in asterile biosafety hood in a separate room away from the main laboratory. That nosequences similar to those reported here have been amplified from DNA sam-ples from other HIV-1-infected chimpanzees during hundreds of PCRs in ourlaboratory indicates that the sequences are unique to these animals.
DNA heteroduplex assay. To assess the diversity of clones generated by PCR,60 clones containing the p17gag gene and 83 clones containing C2-V5env wereselected for analysis by HMA, performed essentially as described by Delwart etal. (12, 13) except that we included 2.7 M urea to stabilize mismatched hetero-duplexes. The HMA was used primarily as a prescreen to ensure that divergentclones were sequenced and that the extent of diversity was as accurate as pos-sible. One clone each from env and gag (clone 6 for C2-V5env and clone 1C forp17gag) was selected at random as a probe to evaluate the other clones forheterogeneity. Plasmid DNA (5 ng) from each cloned fragment or 2.5 ml of thelysed (heated) bacterial culture was amplified again by PCR to provide sufficientproduct, and then equal volumes of the PCR products of the probe and other TAclones were used for the HMA. After denaturation at 95°C and slow cooling ofthe mixtures to room temperature to allow reannealing, aliquots of the reactionswere electrophoresed on 5% polyacrylamide gels containing 2.7 M urea. Gelswere stained with ethidium bromide, and heteroduplexes were visualized underUV light.
DNA sequencing and phylogenetic analysis. Nucleotide sequences of frag-ments cloned from plasmid DNA were determined by the standard dide-oxynucleotide chain termination method, using Sequenase version 2 (U.S.Biochemicals, Cleveland, Ohio) according to the manufacturer’s protocol.For sequencing p17gag and C2-V5env, the two inner PCR primers were used tosequence in both forward and reverse directions. In the gag gene, an overlap ofapproximately 50 bp was read. One internal primer was required for env; thisprimer was B3A (59-GCACAGTTTTAATTGTGGAG-39 [bases 7342 to 7361 inthe HXB2 molecular clone]), and 10- to 20-base overlaps were generated. Forthe nef gene, three forward primers, which allowed overlaps of approximately20 bases to be read, were used. These primers, also based on the HXB2 molec-ular clone, were nef0 (59-CTTGGAAAGGATTTTGCTATA-39 [bases 8773 to8793]), nef5 (59-TGGCTAGAAGCACAAGAGGA-39 [bases 8964 to 8983]),and nef7 (59-AGCTAGTACCAGTTGAGCCA-39 [bases 9226 to 9245]).
FIG. 1. Inoculation history of chimpanzee C-499 (24). The asterisk indicates the time at which blood was obtained from C-499 for the studies reported here. Thevirus identified as HIV-1JC by Novembre et al. (54) was obtained, as indicated, when C-499 had clinical AIDS.
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The parental HIV-1LAI sequences were used for all analyses because DNAsequences of 10 clones of the region surrounding the V3 loop of the LAV-1bvirus stock were identical to those of the original isolate (data not shown),demonstrating limited diversification during the 5 months of passage in C-459before this stock was prepared. Translation to amino acids and amino acidsequence alignments were done with MacVector version 5.0 (Eastman KodakCo., New Haven, Conn.), with minor manual adjustments. Genetic distanceswere determined by pairwise comparisons using the two-parameter method ofKimura, excluding gaps caused by insertions or deletions (PHYLIP package,version 3.572). The neighbor-joining method was used to analyze sequencerelationships and to construct phylogenetic trees, which were evaluated statisti-cally by 100 bootstrap replicates (PHYLIP).
Nucleotide sequence accession numbers. GenBank accession numbers for theenv sequences from C-499 are U56866 to U56887 and AF027771 to AF027785,those for p17gag are U56888 to U56899, and those for nef are AF027786 toAF027806.
RESULTS
Status of chimpanzees. Approximately 1 year before diag-nosis of AIDS and 9 years after initial infection with HIV-1SF2(Fig. 1), peripheral blood was obtained from C-499. At thesame time, blood was collected from another chimpanzee,C-459, that had been infected for more than 10 years withHIV-1LAI(LAV); C-459 was the animal through which theLAI(LAV) strain had been passaged for 5 months before theLAV-1b virus stock that was used to inoculate C-499 wasgenerated (21). Also included in the study was chimpanzeeC-487, which had been infected for 9 years with an aliquot ofthe same HIV-1LAV-1b stock with which C-499 was inoculated.At the time blood was collected from each animal, anti-HIV-1antibody titers, viral burdens, and percentages of CD41 andCD81 T cells were determined (Table 1). C-487 had the high-est levels of both antibody titers and copies of virion RNA inplasma. C-499 also had high antibody titers and a CD4/CD8ratio of 0.24, which was comparable to that of C-487. TheCD4/CD8 ratio for the third animal, C-459, was normal. At thetime this analysis was done, HIV-1 was isolated from PBMCfrom C-499 and C-487 but not from C-459. Serial dilutions ofpurified CD41 lymphocytes from C-499 indicated that 104
CD41 T lymphocytes contained a minimum of one infectiouscell.
Genetic analysis of p17gag. For p17gag, one of the most vari-able proteins encoded in HIV-1 gag (44), HMA of 17 clonesPCR-amplified from approximately 1.5 3 105 unculturedPBMC from C-499 revealed nine different heteroduplex band-ing patterns (Fig. 2), with six patterns represented by one cloneeach. PCR amplification of p17gag from cocultured PBMC fol-lowed by HMA resulted in seven HMA patterns, three ofwhich were not represented by clones from uncultured PBMC(Fig. 2, lanes 1C, 38C, and 36C). (Note that clones from cul-tured PBMC are indicated by a C after the clone number.)That the 12 HMA patterns identified in the 53 p17gag clonesanalyzed reflected the true extent of diversity is supported bydata obtained from the analysis of the C2-V5env region, whichindicated that at least 36 distinct proviruses were present (see
below). Interestingly, the HMA pattern represented by themost clones (6 of 17 clones, or 35%) from uncultured PBMCalso was most prevalent (17 of 36 clones, or 47%) among thosefrom cultured PBMC (represented by clone 39C). Since themost diverse HMA pattern (Fig. 2, lane 8), relative to theprobe, was represented by clones from both cultured and un-cultured PBMC, the extents of diversity in the two PBMCpopulations were similar. Nucleotide sequencing of two cloneswith the same HMA pattern revealed that the difference be-tween these two clones was less than the nucleotide differencesbetween clones with different patterns; for example, clone 39Cdiffered from the probe, clone 1C, by only 2 bp (Fig. 2). Forthis reason, only one clone representing each pattern was se-lected for nucleotide sequence analysis.
Pairwise comparisons of each DNA sequence with all others,including those of the original infecting viruses, revealed thatthe percent nucleotide distances of the 12 p17gag sequencesrelative to SF2 and LAI were equivalent (Table 2); the intra-clone distance ranged from 0.45 to 6.3%. (The nucleotidedifference between p17gag encoded in the parental HIV-1strains SF2 and LAI is 4.17%.) Although these comparisonsdid not allow the parentage of the clones, that is, whether theywere derived from the SF2 or LAV-1b strain, to be deter-
FIG. 2. HMA of PCR amplicons of the p17gag gene from PBMC of C-499.Clone 1C was chosen at random and used as the probe; the first lane is the 1Chomoduplex control. Each lane represents heteroduplexes of individual cloneswith 1C, ordered by increasing diversity. Clone numbers followed by a “C”indicate amplification from cultured PBMC, whereas numbers alone identifyclones from uncultured PBMC. The top of the figure is coincident with the topof the gel.
TABLE 1. Status of chimpanzees at time of genetic analysis
a HIV-1-specific antibody titers were defined as the reciprocal of the last serum dilution giving an optical density reading above the cutoff value and were determinedwith Genetic Systems LAV EIA kit (Sanofi Diagnostics Pasteur, Seattle, Wash.) according to the manufacturer’s instructions.
b The number of copies of HIV-1 genomic RNA in plasma was determined with the Amplicor HIV Monitor kit, a quantitative reverse transcription-PCR assay,according to the manufacturer’s instructions (Roche Diagnostic Systems, Branchburg, N.J.).
c Values are likely to be below the actual number of copies/milliliter because the plasma samples contained heparin which can interfere with the assay.
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mined, all of the clones contained a 6-bp (two-amino-acid)insertion near the 39 end of the p17gag gene in the SF2 strain(Fig. 3). These six bases are not in LAI and are rarely found inother subtype B strains (53). In an attempt to clarify the rela-tionships, a phylogenetic tree of the p17gag clones was con-structed (Fig. 4). Included in the analysis were sequences fromHIV-1 strains LAI and SF2, seven clade B strains chosen atrandom from the database (53), and the clade D strain NDK,to which C-499 also was exposed. Although all of the p17gag
sequences branched with the HIV-1LAI strain, the bootstrapvalue for 100 trees was only 26; therefore, the parental viruscould not be identified with certainty.
Genetic analysis of C2-V5env. Because the diversity in envgenes is generally greater than that in gag, 30 TA clones,generated by PCR from proviral DNA isolated from uncul-tured PBMC, were analyzed by HMA to estimate the extent ofgenetic diversity in C2-V5env. HMA of these 30 clones revealed21 distinct patterns; 16 patterns were represented by only oneclone, four patterns were represented by two clones, and themost prevalent group was represented by five clones (24%),indicating that the majority of PCR products were amplifiedfrom different proviruses. Furthermore, these HIV-1 provi-ruses in C-499’s uncultured PBMC formed two distinct popu-lations: the larger population was represented by 21 clones(70%), and the smaller population was represented by 9 clones(30%). Proviral DNA was also isolated from C-499’s PBMCthat had been cocultured with normal human PBMC for 15days; all 53 clones from two independent PCRs were related to
the major population of clones identified among proviruses inthe uncultured PBMC. In contrast to the clones from uncul-tured PBMC, 19 (36%) of the 53 clones from cultured PBMChad essentially the same HMA pattern, indicating that onevariant (represented by one clone among the unculturedPBMC PCR products) predominated during culture (Fig. 5,clone 3c). However, 14 additional HMA patterns not identifiedamong the uncultured PBMC were observed after culture; 12of these 14 patterns were represented by a single clone. Thatmost HMA patterns from both uncultured and cultured PBMCwere represented by only one or two clones illustrates theextreme diversity of C-499’s quasispecies and that most cloneswere derived independently.
When all 83 clones from uncultured and cultured PBMCwere compared by HMA, the major group was represented by29 different banding patterns (Fig. 5), while seven patternswere identified for the minor group. Analyses of nucleotideand predicted amino acid sequences of the C2-V5env fragmentsconfirmed that clones in the most prevalent group were de-rived from HIV-1LAV-1b, whereas the more divergent minorpopulation of seven clones appeared to contain regions similarto both HIV-1LAV-1b and HIV-1SF2 (Fig. 6; see below). Theextent of env divergence of all clones from either parentalstrain was significantly greater than that observed for thep17gag gene (Table 2). This extreme diversity, which was com-parable to that documented by Novembre et al. (54) in the V1and V2 env regions of HIV-1JC, resulted not only from nucle-otide substitutions but also from insertions and deletions,many of which were localized within the V4 and V5 regions ofgp120env. Only four clones appeared to be defective because ofpremature stop codons in V3 (clone II.32C) or V4 (clones 26,24, and 18).
A phylogenetic tree was constructed with the sequences gen-erated in this study, the three HIV-1 strains to which C-499 wasexposed (LAI, SF2, and NDK), seven clade B strains chosen atrandom from the database (53), a clade A strain, and a cladeC strain, to which the tree was rooted (Fig. 7). This phyloge-netic analysis not only confirmed that two distinct env popula-tions, the minor group of which formed a monophyletic cluster,were present in C-499’s PBMC but also suggested that bothgroups were derived from HIV-1LAV-1b; however, the boot-strap value for this node was 40, indicating a weak association.That clones from the cultured PBMC were interspersed amongthose in the major population from the uncultured PBMCindicated that multiple diverse genotypes may have been rep-lication competent. When the sequences of the minor popula-tion were compared pairwise to LAI and SF2, the genetic
FIG. 3. Nucleotide (A) and amino acid (B) sequences of the 39 termini of the p17gag genes of HIV-1SF2, HIV-1LAI, and representative clones from C-499. Dashesindicate identity with HIV-1SF2, and dots indicate deletions. The six base pairs (two amino acids) in HIV-1SF2 that are not present in HIV-1LAI are underlined.
TABLE 2. Genetic distances between HIV-1JC499 and the parentalstrains, LAI(LAV) and SF2
Gene GroupaGenetic distance (%)
LAI SF2 Intraclone
p17gag All 3.11–8.3 4.63–9.29 0.45–6.3C2-V5env All 9.54–15.2 9.37–20.2 0.78–19.28
Minor 9.54–11.47 9.37–11.4 1.4–4.48Major 11.98–15.2 16.13–20.2 0.78–8.47
59-C2-V3 Minor 12.6–15.33 6.1–9.42Major 13.25–15.56 18.52–21.91
V4-V5-39 Minor 7.4–9.42 11.6–14.92Major 9.74–15.55 12.93–20.25
nef All 6.33–7.9 4.02–5.55 0.16–3.81
a With respect to whether all clones or, in the case of C2-V5env, only a subsetof clones were analyzed. Minor, the C2-V5env group represented by the smallernumber of clones; major, the larger group of clones (Fig. 6).
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distances from the two progenitor strains were essentially thesame (Table 2), which did not allow a distinction of parentage.Similar pairwise comparisons of only the major group of C2-V5env clones, however, indicated that they were probably de-rived from LAV-1b.
To determine whether the extreme degree of diversificationin C2-V5env of HIV-1SF2 and HIV-1LAV-1b during 9 years ofinfection of C-499 was representative of that in other chim-panzees infected with HIV-1LAI-derived strains for compara-ble times, this region of proviral DNA in PBMC from chim-panzees C-459 (10 years) and C-487 (9 years) was analyzed.For each animal, clones from two independent PCR amplifi-
cations of proviral DNA were sequenced (Fig. 8). Among sixclones from C-459’s proviral DNA, the intraisolate nucleotidedistances ranged from 0.45 to 3.76%, whereas pairwise differ-ences with HIV-1LAI ranged from 1.82 to 2.48%. For C-487,the differences among eight clones ranged from 0.93 to 7.51%;pairwise distances from HIV-1LAI ranged from 4.1 to 9.65%.Similar to the low frequency of apparently defective clonesfrom C-499, none of six and only one of eight clones fromC-459 and C-487, respectively, appeared to encode a defectiveEnv protein. That there was greater diversity among clonesfrom C-487 than those from C-459 is consistent with the his-tories of these two chimpanzees and the higher viral burden inC-487, which is reflected, in part, by this animal’s high HIV-1-specific antibody titer (Table 1). In addition, virus was iso-lated from C-487’s PBMC on 100% of attempts throughout its9 years of infection, which was not true for similar virus isola-tion attempts from C-459. Furthermore, DNA sequence andphylogenetic analyses of the clones obtained from C-459 re-vealed two distinct populations of closely related sequences(Fig. 9). However, the intraisolate pairwise distances are wellabove the error frequency for Taq polymerase (,0.05%), sug-gesting that most clones were derived from different provi-ruses. Although not measured directly because of insufficientsample, it is likely that the proviral copy number in C-459’sPBMC was probably substantially lower than that in C-487’sPBMC. Serial dilution of C-487’s genomic DNA and subse-quent PCR resulted in amplification of HIV-1 sequences at a1:32 dilution, and the products of this reaction had four distinctpatterns on HMA gels (data not shown). These results indi-cated that the minimum number of proviruses in 1 mg of DNAfrom C-487’s PBMC (;1.5 3 105 cells) was between 32 and128 (within the limitations of the assay), supporting the con-clusion that the clones more than likely did not arise fromamplification of one or only a few proviruses.
Evidence for recombination. As discussed above, the minorpopulation of C2-V5env sequences from C-499 appeared tobranch with HIV-1LAV-1b (Fig. 7); however, visual comparisonof the amino acid sequences of the V3 loops strongly indicatedthat this minor population was derived from HIV-1SF2 (Fig. 6).The ambiguity in the parentage of the minor population sug-gested that this group was comprised of recombinant viruses.
FIG. 4. Phylogenetic relationship of the p17gag nucleotide sequences of HIV-1LAI and HIV-1SF2 to clones derived from proviral DNA in cultured (denoted bya “C”) and uncultured PBMC from chimpanzee C-499. The tree was rooted tothe clade D strain NDK and includes seven HIV-1 clade B strains chosen atrandom from the database (53). Only bootstrap values greater than 50 are shownat nodes, unless the text refers to specific nodes with lower bootstrap values.Horizontal branch lengths reflect the genetic distance between sequences.
FIG. 5. Heteroduplex mobility assay of PCR amplicons of C2-V5env from cultured and uncultured PBMC of C-499. Clone 6 was chosen at random and used as theprobe; the first lane shows the clone 6 homoduplex control. Each lane represents heteroduplexes of clone 6 with individual clones, ordered by increasing diversity. Twodistinct populations were identified; the major and minor groups are seen in the first 29 lanes and the last 7 lanes, respectively. The heteroduplex banding pattern forclone 18 reflects a deletion in this region.
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To test this possibility, we evaluated separately the 59 half ofthe C2-V5env fragment, which included C2, V3, and part of C3(59-C2-V3), and the 39 half, which included part of C3, V4, C4,and V5 (V4-V5-39). Phylogenetic trees of these two subfrag-
ments confirmed that the region encompassing the V3 loop ofthe minor and major populations were derived from SF2 andLAV-1b, respectively (Fig. 10A). In the V4-V5-39 region, thetwo populations, while still distinct, did not branch with either
FIG. 6. Amino acid sequence alignment of C2-V5env clones from C-499’s cultured and uncultured PBMC with HIV-1SF2 and HIV-1LAI. Dashes indicate identitywith the parental strains, and dots signify gaps. Clone numbers preceded by “I” or “II” identify clones from two independent PCRs. The positions of V3, V4, and V5are underlined and delineated by the arrows above cysteine residues. p, stop codon.
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parental strain (Fig. 10B); however, a comparison of the pair-wise distances from SF2 and LAV-1b revealed that most clonesin both populations were more closely related to LAV-1b thanSF2 and that, more importantly, the smaller population wasclearly more closely related to LAV-1b than to SF2 (Table 2).
Genetic analysis of nef. Because Nef appears to be importantfor pathogenicity in the simian immunodeficiency virus-ma-caque model (40), the entire nef gene in proviral DNA fromC-499’s cultured PBMC was PCR amplified, and multipleclones from two reactions were sequenced. Of the 21 clonessequenced, only 3 (14.3%) were defective, and all of theseencoded premature stop codons. The charged acidic motif atamino acids 62 to 65 (EEEE), the SH3-binding motif [(PXX)4]at amino acids 69 to 80, and the b-turn motif (GPGI) at aminoacids 130 to 133 were conserved in all full-length clones (47,66). Pairwise differences for all nef clones revealed a closersequence relationship to the SF2 strain, and all of the nefclones branched with the SF2 strain in the phylogenetic tree(Fig. 11). However, none of these clones contained a 12-bp(four-amino-acid) insertion near the 59 end of HIV-1SF2 that isnot found in HIV-1LAI (53).
DISCUSSION
The results of this study show that during 9 years of infectionof a chimpanzee with two HIV-1 subtype B strains, extensivediversification occurred in C2-V5env and, to a lesser extent, inp17gag and nef. The divergence from the parental strains in envwas greater than that for p17gag and nef, suggesting that Envhad evolved independently from Gag and Nef during the 9years of infection, which is consistent with observations duringHIV-1 infection of humans (11, 46). Furthermore, in the C2-V5env clones generated from proviral DNA in uncultured
FIG. 7. Phylogenetic relationship of C2-V5env clones from C-499 to the pa-rental strains and randomly chosen strains from clade B and clades A (Z321), D(NDK), and C (NOF), to which the tree is rooted. Clones designated by numbersonly are from uncultured PBMC; the remaining clones are from two independentPCRs (I and II) of cultured (C) PBMC. See the legend to Fig. 4 for otherexplanations.
FIG. 6—Continued.
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PBMC, there were two distinct populations of related ge-nomes, with one population more prevalent than the other(approximate ratio, 3:1). The 59-C2-V3 region of the minorpopulation clearly was derived from HIV-1SF2, and the majorpopulation was derived from HIV-1LAV-1b. Since both the ge-netic distances and the phylogenetic tree indicated that in theV4-V5-39 region of Env, the minor population was moreclosely related to HIV-1LAV-1b, these results support the con-clusion that the quasispecies in the minor population are re-combinant genomes in C2-V5env. No evidence for sequencesrelated to HIV-1NDK was obtained. Identifying the progenitorsof p17gag and nef was more problematic. If one considers onlythe phylogenetic trees, then p17gag was derived from LAV-1band nef was derived from SF2; however, the nucleotide andamino acid sequences of p17gag have features unique to SF2,and those of nef have features unique to LAV-1b. If eitherp17gag or nef was derived from SF2, then additional recombi-national events may have occurred. To verify this, however, willrequire sequencing a molecularly cloned virus (or viruses)from C-499’s PBMC and performing a breakpoint analysisthroughout the entire genome (60).
Analysis of the Env V3 loop. In the V3 loop there weresubstantially more amino acid changes in proviruses derivedfrom HIV-1LAV-1b than in those that evolved from HIV-1SF2.In the majority of V3 loop amino acid sequences in the LAV-1b-derived group, more than one-third (36%) of the amino
acids had changed, compared with only 20% for the SF2-derived group. This latter percentage was comparable to the19% amino acid differences in the V3 loop for C-487. Similarly,proviral DNA from a laboratory worker infected with theclosely related HIV-1LAI(IIIB) strain for 5 years had accumu-lated only 11% diversity (59). If one compares these percentchanges with those in unrelated strains, the amino acid differ-ence in the V3 loop between viruses present in an HIV-in-fected hemophiliac at seroconversion and 7 years later was11.4% (36). These observations indicate that greater diversifi-cation had occurred in C-499’s quasispecies with the LAV-1b-related V3 loop; furthermore, one of these changes was un-usual. Although the original GPGR motifs in the tip of theloops of both parental strains had accumulated mutations, theywere intrinsically different. All HIV-1SF2-derived viruses hadacquired a GPGK motif, which is not uncommon among cladeB isolates. In contrast, all HIV-1LAV-1b-derived clones fromC-499 encoded GYGR, a tetrad not found in any of the 707HIV-1 V3 sequences in the database, irrespective of cladedesignation (53).
Other genetic factors that might influence properties ofHIV-1, such as cell tropism, include the net charge of the V3loop and the number and placement of N-linked glycosylationsites. In C-499’s quasispecies, the number of positively chargedamino acids in the V3 loop of the HIV-1LAV-1b-related viruseshad increased from 9 to 12. Such increases in positive charge
FIG. 8. Amino acid sequence alignment of C2-V5env clones from C-459’s and C-487’s uncultured PBMC with HIV-1LAI. See the legend to Fig. 6.
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tend to increase macrophage tropism of HIV-1 isolates (9, 16,41); however, since the HIV-1LAV-1b stock replicates efficientlyin chimpanzee macrophages (27, 70), the effects of increasedcharge, if any, on this property cannot be predicted.
Analysis of the Env V4-V5 region. In the V4 loop, most ofthe minor population of clones had 15 changes among the 29amino acid residues (52%), which included an insertion of
three and a deletion of two amino acids, whereas most of theclones in the major population had 12 amino acid changes,which included a deletion of 6 of the original 34 amino acids(35%). As observed in the V4 loop consensus sequence forC-487 and one group of clones from C-459, one copy of theFNSTW repeat was deleted; with the exception of three of thesix clones from C-459, this deletion has been found in allisolates from chimpanzees infected with HIV-1LAI-relatedstrains that we have sequenced (17). However, two amino acidsof the second FNSTW in the consensus sequence for C-459’squasispecies are different, suggesting that this repeat motifmay be specifically selected against in chimpanzees. The morelimited evolutionary changes in the quasispecies from the twoasymptomatic chimpanzees were confirmed in the phyloge-netic analysis (Fig. 9) and are consistent with the results ofReitz et al. (59). These investigators reported only 1.2% nu-cleotide divergence in 246 nucleotides encompassing the V4loop in proviral DNA tested 5 years after a laboratoryworker was infected with the closely related HIV-1LAI(IIIB)strain.
The most extensive changes were found in the V5 region ofthe clones from C-499. In a majority of the minor populationof clones, 6 of 10 amino acids were altered, and 50% of theclones had a two-amino-acid insertion and/or a one-amino-aciddeletion. In the major population of clones, 7 of 9 (78%) of theamino acids were altered in combination with various inser-tions and deletions. Consistent with diversification of otherregions of C2-V5env, the quasispecies from C-459 had no oronly two altered amino acids, while the frequency of mutationin V5 of proviral genomes from C-487 were intermediate tothat of C-459 and C-499, resulting in changes in 4 of 9 (44%)of the V5 amino acids, with two others deleted in all but oneclone. Thus, the concentration of mutations in the V3, V4, andV5 regions of all clones from C-499 and C-487 indicates thatthese variable regions were subject to considerable selectivepressure in these two long-term HIV-1-infected chimpanzees.
FIG. 9. Phylogenetic relationships of C2-V5env clones from C-459 and C-487to the parental HIV-1LAI. See legends to Fig. 4 and 7 for other explanations.
FIG. 10. Phylogenetic trees of the 59-C2-V3env (A) and V4-V5env-39 (B) fragments of clones from C-499. See legends to Fig. 4 and 7.
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Analysis of Nef. As previously noted in HIV-1 quasispeciesin humans (11, 37, 58, 66), other than multiple amino acidchanges in an area of sequence polymorphism near the 59terminus of C-499’s nef genes, other mutations resulting inamino acid changes were scattered at random throughout Nef.This finding and the overall lower diversity in Nef, compared tothat in the p17 Gag and Env proteins, suggest there is less (orno) selective pressure on the nef gene, which is consistent withevaluations of nef gene evolution over time in HIV-1-infectedhumans (11, 58). In fact, Plikat et al. (58) concluded thatgenetic drift was the major factor in evolution of nef andpredicted that after 10 years of infection, a nef quasispecieswould diverge 7.1%. The divergence of C-499’s nef sequencesfrom both HIV-1SF2 and HIV-1LAV-1b is in remarkable agree-ment with this estimate (Table 2).
Diversification and disease. While some studies of virusevolution in HIV-1-infected humans have concluded that dis-ease progression is associated with more extensive genetic di-versity (55, 65, 67), others have shown either that diversitycorrelates with longer asymptomatic periods (7, 12, 26, 43, 45,48, 69, 72) or that there is no correlation (2, 71, 74). In onestudy, pairwise comparisons of the V3 consensus nucleotidesequences of isolates from six children infected by blood prod-ucts from a single donor indicated that the intraperson varia-tion (range, 0.3 to 2.9%) was similar to that observed in C-459’sproviruses (71). The aforementioned study also revealed nocorrelation between genetic heterogeneity and disease, butrelative to the donor inoculum, the progressors tended to har-bor viral genomes with less divergence than the nonprogres-sors. In a similar evaluation of six hemophiliacs who receivedfactor VIII from the same donor, after 5 years the interpatientvariation in the V4-V5 region ranged from 5.6 to 11.1%, but arelationship between intrapatient HIV-1 diversity and viralburden in PBMC was not observed (2). More recently, ananalysis of nonsynonymous mutations in the V3 region of
HIV-1 in 44 persons evaluated at seroconversion and 5 yearslater indicated that intrahost evolution was directly related tothe duration of the immunocompetent period (45). This con-clusion was supported by Wolinsky et al. (72), who found aninverse correlation between rapid disease progression and bothgenetic diversity in the V3-V5 region and the frequency ofcytotoxic T-lymphocyte precursors. Most studies relate diver-sity in various regions of env to disease progression; however,Yoshimura et al. (73) evaluated the full-length gag gene andfound that the extent of variation appeared to correlate withduration of infection.
In contrast to many studies of HIV-1 infections in humans,the genetic diversity was much greater in the HIV-1 provirusesin PBMC from chimpanzee C-499, which developed AIDS,than in PBMC from the other two chimpanzees that wereinfected for a comparable time but did not develop clinicaldisease. It is interesting, however, that C-487, with a level ofdiversification intermediate between that of C-459 and C-499,had experienced repeated stimulation of the immune systemduring the initial 2 years of infection, each episode of whichwas accompanied by transient increases in HIV-1 viral burdenin peripheral blood (19). These manipulations may have con-tributed to maintenance of high viral loads and evidence ofimmune dysfunction—16.5% CD41 lymphocytes, a CD4/CD8ratio of 0.23 (Table 1), and elevated levels of apoptotic lym-phocytes compared to normal chimpanzees (10). However,whether the observed genetic diversification contributed to orwas a consequence of disease progression cannot be deter-mined. Thus, no consistent correlation between genetic diver-sification in env and disease progression has emerged in co-horts of humans (or chimpanzees) infected with the samestrain. It is likely that major factors in the degree of diversifi-cation and disease progression are virus-host interactions spe-cific to each individual and the HIV-1 strain that they harbor(74).
The observed difference in diversification in the three chim-panzees cannot be explained by length of infection becauseC-459 had been infected longer than C-499. Likewise, it cannotbe explained by viral burdens over the course of infection or atthe time these blood samples were obtained (Table 1), which isconsistent with a study of humans by Balfe et al. (2). DespiteC-487’s extremely high HIV-1-specific antibody titers, whichare directly proportional to viral burden in chimpanzees (29,39), during the past 9 years virus has been isolated from itsPBMC on every attempt using standard culture conditions, andfor the last 2 to 3 years, this animal has maintained a level ofvirus between 2 3 103 and 1.3 3 104 RNA copies/ml of plasma.These quantities of virus are comparable to those in a group ofpatients who developed AIDS between 6 and 9 years afterinfection, described by Henrard et al. (35). The 100% successat isolating HIV-1 from C-487’s PBMC was not observed withC-459 or C-499 before evidence of disease developed. The lowHIV-1 RNA copy number (724 copies/ml) that we found inplasma from C-499 may be an underestimate because the onlyplasma sample available contained heparin and had been fro-zen for almost 2 years. Using a different assay, Novembre et al.(54) found approximately 105 RNA equivalents/ml of plasmaduring the last few months before C-499 was euthanized. Thevalues obtained in that and the present study indicate thatsome HIV-1-infected chimpanzees have levels of virion RNAin plasma comparable to that in humans (35, 49, 50). In addi-tion, it is possible that the extreme genetic diversity in C-499’squasispecies resulted from, or was influenced by, the long-termcoexistence of two distinct HIV-1 strains that together en-hanced the degree of evolution driven by immune selection.Because there are now several chimpanzees coinfected with
FIG. 11. Phylogenetic relationship of nucleotide sequences of nef genes fromC-499’s cultured PBMC to those of HIV-1SF2 and HIV-1LAI. The tree was rootedto NDK. See legends to Fig. 4 and 7.
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two different strains of HIV-1, it will be possible to evaluate theinfluence of coinfection on genetic diversity.
An outcome of the present study was the identification ofrecombinant proviruses between HIV-1SF2 and HIV-1LAV-1bin C2-V5env and indications that additional recombinatorialevents may have occurred. In one aspect this was surprisingbecause, in comparison to HIV-1LAV-1b, HIV-1SF2 not onlyreplicates poorly in chimpanzee PBMC in vitro but also estab-lishes significantly lower viral burdens in infected animals andcan rarely be isolated from PBMC after the first 2 months ofinfection (24, 29, 52). Since HIV-1SF2 was inoculated 15months before HIV-1LAV-1b, then HIV-1SF2 had to be activelyreplicating in some body compartment so that individual cellswere infected with both strains. It appears that recombinationbetween distinct HIV-1 strains in dually infected chimpanzeesmay be as prevalent as it appears to be in humans (14, 42, 60,61). We recently reported the identification of chimeric sub-type B and E env genes in lymph node tissue obtained 24 weeksafter a chimpanzee had been coinfected with HIV-1LAI(IIIB)and HIV-190CR402 (25). Furthermore, we subsequently ob-tained evidence of interclade recombination in another duallyinfected chimpanzee (17). Thus, the HIV-chimpanzee modelmay provide a way to document the timing and extent ofrecombination that can occur between two (or more) HIV-1strains during coinfections.
Although the inherent pathogenicity of HIV-1 strains mayinfluence disease progression, since all three chimpanzeeswere infected with the same strain, this factor should not havebeen important. The original HIV-1LAV-1b inoculum thatC-499 received has an SI phenotype, is cytopathic for chim-panzee PBMC, and is both macrophage and T-cell tropic; itnot only replicates well in chimpanzee bone marrow- andblood monocyte-derived macrophages but also induces syncy-tia in both MT2 cells and chimpanzee and human PBMC (27,70). Although these properties may have contributed to dis-ease progression, they are unlikely to be the determining fac-tors because C-487 was inoculated with the same virus stock. Inpreliminary in vitro studies, the quasispecies recovered fromC-499 has retained its SI phenotype for both MT2 cells andhuman PBMC, which is consistent with the results of Novem-bre et al. for HIV-1JC (54). It should be noted, however, thatNovembre et al. (54) were incorrect in their statement thatC-499 was not inoculated with a strain of HIV-1 that formssyncytia in chimpanzee PBMC. The cytopathic effects of HIV-1LAV-1b for chimpanzee cells, including syncytium formation,have been well documented (21, 27, 70). Comparisons of bio-logic and molecular properties of HIV-1 recovered from theseand other long-term-infected chimpanzees with and withoutevidence of disease may provide insight into determinants ofHIV-1 pathogenesis.
A logical extension of the characterization of this heteroge-neous quasispecies from C-499 is to determine whether cell-free HIV-1 recovered from this chimpanzee that developedAIDS is pathogenic for other chimpanzees. Although Novem-bre et al. (54) reported a rapid decline in a chimpanzee inoc-ulated with C-499’s virus, that animal was the recipient of atransfusion of 40 ml of whole blood, and therefore it receivedan extremely high dose of virus. We have inoculated two chim-panzees, one intravenously and one by atraumatic exposure tothe cervical mucosa, with cell-free supernatant from a cocul-ture of C-499’s PBMC with normal human PBMC; this virusstock established persistent high viral burdens in both chim-panzees. In addition, during 21 months of infection, both an-imals have exhibited steady declines in percentages and num-bers of CD41 lymphocytes (10). This same combination ofhigh levels of plasma RNA and decreasing numbers of CD41
cells is predictive of disease progression in humans (49, 50, 56).Thus, that chimpanzees can exhibit pathogenic sequelae as aresult of HIV-1 infection validates their continued use to eval-uate the potential efficacy of HIV-1 vaccines. The use of vi-ruses, such as the one described here (which we call HIV-1JC499), will allow investigators to assess vaccine-mediatedprotection not only against infection but also against disease.
ACKNOWLEDGMENTS
We thank Jackie Stallworth for technical assistance, Dawn Grill forsecretarial assistance, Beatrice Hahn for suggestions on the geneticanalysis, and Harold McClure, Yerkes Primate Research Center, forproviding blood samples from C-459 and C-499.
This work was supported in part by NIH grant AI28147 and PasteurMerieux Serum and Vaccins.
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