PML plays both inimical and beneficial roles inHSV-1 replicationPei Xua, Stephen Mallona, and Bernard Roizmana,1

aMarjorie B. Kovler Viral Oncology Labs, The University of Chicago, Chicago IL 60637

Contributed by Bernard Roizman, April 15, 2016 (sent for review January 21, 2016; reviewed by David C. Bloom and Rozanne M. Sandri-Goldin)

After entry into the nucleus, herpes simplex virus (HSV) DNA is coatedwith repressive proteins and becomes the site of assembly of nucleardomain 10 (ND10) bodies. These small (0.1–1 μM) nuclear structurescontain both constant [e.g., promyelocytic leukemia protein (PML),Sp100, death-domain associated protein (Daxx), and so forth] andvariable proteins, depending on the function of the cells or the stressto which they are exposed. The amounts of PML and the number ofND10 structures increase in cells exposed to IFN-β. On initiation ofHSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML andSp100. The earlier report that IFN-β is significantly more effective inblocking viral replication in murine PML+/+ cells than in sibling PML−/−

cells, reproduced here with human cells, suggests that PML acts as aneffector of antiviral effects of IFN-β. To define more precisely thefunction of PML in HSV-1 replication, we constructed a PML−/− humancell line. We report that in PML−/− cells, Sp100 degradation is delayed,possibly because colocalization and merger of ICP0 with nuclear bod-ies containing Sp100 and Daxx is ineffective, and that HSV-1 replicatesequally well in parental HEp-2 and PML−/− cells infected at 5 pfu wild-type virus per cell, but poorly in PML−/− cells exposed to 0.1 pfu percell. Finally, ICP0 accumulation is reduced in PML−/− infected at low,but not high, multiplicities of infection. In essence, the very mecha-nism that serves to degrade an antiviral IFN-β effector is exploited byHSV-1 to establish an efficient replication domain in the nucleus.

ND10 | ICP0 | Sp100 | Daxx | interferon

Several prominent events take place after the entry of herpessimplex virus (HSV) DNA into the nucleus of newly infected

cells. Thus, viral DNA becomes coated by repressive proteins, thefunction of which is to block viral gene expression (1–6); nucleardomain 10 (ND10) bodies colocalize with the viral DNA (7, 8); α orimmediate early viral genes are expressed; and one viral protein,ICP0, degrades promyelocytic leukemia protein (PML) and Sp100,two key constituents of ND10 bodies in conjunction with theUbcH5A ubiquitin-conjugating enzyme (9–11). What is left of theND10 bodies is infiltrated by viral proteins and becomes the viralreplication compartment (12–15).ND10 bodies range between 0.1 and 1 μM in diameter. The

composition of ND10 bodies varies depending on the cellularfunction or in response to stress, such as that resulting from virusinfection (16–19). Among the constant components of ND10 arePML, Sp100, and death-domain associated protein (Daxx). PMLhas been reported to be critical for the recruitment of componentsand for the organization of the ND10 bodies (18–23). The functionof ND10 bodies may vary under different cellular conditions andmay also depend on their composition.A key question that remains unanswered is the function of ND10

bodies in infection, and in particular, why HSV has evolved astrategy that specifically targets PML and Sp100 for degradation.Two clues that may ultimately shed light on the function of ND10 isthat exposure of cells to IFN leads to an increase in the number ofND10 bodies and an increase in PML (16, 24–26). The second clueemerged from the observation reported earlier by this laboratory isthat pretreatment of murine PML+/+ cells with IFN-β led to a drasticreduction in virus yields. In contrast, exposure of PML−/− cells toIFN-β led to a significantly smaller decrease in virus yields (27). The

results suggest PML is an antiviral effector of IFN-β, but manyquestions regarding the function of PML remain unanswered (28).In this study, we constructed a PML−/− cell line (1D2) derived

from HEp-2 cells. The first part of this report centers on thestructure of ND10 bodies bereft of PML and the interaction of thesebodies with ICP0. In the second part, we report on the replication ofHSV-1 in PML−/− cells. Here we show that HSV-1 replication andthe accumulation of ICP0 are significantly reduced in PML−/− cellsexposed to low ratios of virus per cell. HSV has evolved a strategy totake advantage of PML before its degradation.

ResultsGeneration and Properties of PML−/− 1D2 Clone Derived from HEp-2Cells. PML is a family of seven isoforms. The largest, PML I, consistsof nine exons (29–31). PML−/− cell clones were generated fromHEp-2 cells by transfection of clustered regularly interspaced shortpalindromic repeats [clustered regularly interspaced short palin-dromic repeats (CRISPR)/cas9 cassette] targeting exon 1 of PML(32–34). The procedure for drug selection and flow cytometry wereboth performed according to the manufacturer’s instructions and arebriefly outlined in Materials and Methods. Of the numerous clonesderived from single cells, we selected clone ID2 for further study.Two series of experiments were done to verify the absence of

PML in 1D2 cells. In the first, we took advantage of earlier studiesshowing that PML is amplified in cells exposed to IFN-β (26).Specifically, after overnight exposure to IFN-β, extracts of isolatednuclei or total cell lysates were solubilized, subjected to electro-phoresis in a denaturing polyacrylamide gel, transferred to a ni-trocellulose sheet, and reacted with rabbit polyclonal anti-PMLantibody. As shown in Fig. 1A, the antibody to PML reacted withmultiple protein bands present in HEp-2 cells. The antibody didnot react with proteins present in lysates of 1D2 cells.In the second series of experiments, parental HEp-2 or ID2 cells

grown on four-well slides and exposed overnight to IFN-β werereacted with mouse monoclonal antibody to PML (Fig. 1 B, a andb) or with rabbit polyclonal antibody to PML (Fig. 1 B, c and d).HEp-2 cells exhibited typical intranuclear clusters of ND10 bodiescontaining PML. No such structures were observed in 1D2 cells.

Significance

Promyelocytic leukemia protein (PML) is a component of nucleardomain 10 (ND10) bodies and an antiviral effector of IFN-β. Aherpes simplex virus (HSV) protein, ICP0, interacts with PML,merges with ND10 bodies, degrades PML, and ultimately takesover the domain of ND10 bodies. Here we show that viral geneexpression and growth are reduced in PML−/− cells infected atlow ratios of virus to cells. In essence, the results indicate thatHSV-1 evolved themeans to take advantage of an inimical cellulardefense mechanism to degrade it and at the same time use it togain access to a nuclear domain essential for efficient replication.

Author contributions: P.X. and B.R. designed research; P.X. and S.M. performed research;P.X., S.M., and B.R. analyzed data; and P.X. and B.R. wrote the paper.

Reviewers: D.C.B., University of Florida; and R.M.S.-G., University of California, Irvine.

The authors declare no conflict of interest.1To whom correspondence should be addressed. Email: bernard.roizman@bsd.uchicago.edu.

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We did note that ID2 cells contained a ring of green fluorescentpuncta observed in cells reacted with the mouse monoclonal anti-body. These rings of fluorescent puncta in the perinuclear spacewere also present in parental HEp-2 cells but were overshadowedby the ND10 bodies. The presence of the fluorescent puncta inboth HEp-2 and 1D2 cells suggests they are not derived fromtruncated PML proteins. Moreover, the observation that thesepuncta were observed in cells reacted with mouse monoclonalantibody, but not with the rabbit polyclonal antibody against PML,reinforces the conclusion that the antigens reacting with themonoclonal antibody are not derived from PML. We conclude thatPML is not expressed in 1D2 cells.

Effect of IFN-β on Accumulation of Sp100 and Daxx mRNAs. In theexperiments described here, we examined the distribution of Sp100and Daxx in parental HEp-2 cells and in the PML−/− 1D2 clone.Here (Fig. 2), we report that exposure of both parental and 1D2mutant cells to IFN-β enhanced the accumulation of Sp100 but hadno significant effect on the accumulation of Daxx in either theparental HEp-2 or 1D2 cells. The procedures used in this study aredescribed in Materials and Methods.

The Intranuclear Distribution of Sp100 and Daxx in Parental HEp-2 andin Clone 1D2 PML−/− Cells. In this series of experiments, parentalHEp-2 or 1D2 cells were grown on four-well slides in the presenceof growth medium alone or medium supplemented with IFN-β(1,000 U/mL). After 24 h, the cells were fixed and reacted withmonoclonal antibody to PML and polyclonal antibody to Sp100(Fig. 3A) or monoclonal antibody to Daxx and polyclonal antibodyto Sp100. The cells were examined with the aid of a Zeiss confocalmicroscope. The results may be summarized as follows. First, in theabsence of IFN-β, the accumulation of Sp100 was weak and largelyperinuclear (Fig. 3 A, b, c, e, f, and B, b, c, e, f). There were fewND10 structures in parental HEp-2 cells reacted with antibody toPML and Sp100 (Fig. 3 A, c), and these were more readily seen incells reacted with antibodies against Daxx and Sp100 (Fig. 3 B, c).As expected, the monoclonal antibody to PML reacted with anantigen localized in the perinuclear space of 1D2 cells (Fig. 3 A,d and e) in close proximity to Sp100. The structures containingSp100 and Daxx in parental HEp-2 cells could not be differentiatedfrom those detected in nuclei of 1D2 cells.

Second, the number of ND10 bodies was consistently higher inHEp-2 cells treated with IFN-β (Fig. 3 A, i and B, i). As shown inthe enlarged boxed area (Fig. 3 B, i, Inset), the intranuclear fluo-rescent structures were relatively uniform in size and containedboth Sp100 and Daxx.Finally, in 1D2 cells treated with IFN-β, the distribution of an-

tigens reacting with anti-PMLmonoclonal antibody (Fig. 3 A, i) wassimilar to that observed in untreated cells (Fig. 3 A, a). In the IFN-βtreated cells, Sp100 and Daxx colocalized in structures that variedin size (Fig. 3 A, k and, l and B, k and l), but on average, they weresignificantly larger than the ND10 bodies observed in parentalHEp-2 treated with IFN-β (Fig. 3 A, l and B, l).The results of the studies presented here suggest that the

colocalization of Sp100 and Daxx is not dependent on thepresence of PML. However, the variability of the structuresthat contain Sp100 and Daxx suggests that the relatively uniformsizes of ND10 structures in parental HEp-2 cells are defined by

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Fig. 1. Confirmation of the absence of PML in 1D2 clone. (A) Parental HEp-2 and 1D2 cell cultures were exposed to IFN-β (1,000 U/mL). After 24 h, the cells wereharvested, and lysates of nuclei or of whole cells were subjected to electrophoresis in denaturing gels transferred to a nitrocellulose sheet and reacted with rabbitpolyclonal antibody against PML. (B) HEp-2 or 1D2 cells grown on four-well slides were fixed and reacted with mouse monoclonal antibody to PML (a and b) orrabbit polyclonal antibody to PML (c and d). (Magnification: 100×.)

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Fig. 2. Sp100 and Daxx respond differently to exposure of HEp-2 or 1D2 cellsto IFN-β. Parental HEp-2 PML+/+ cells or PML−/− 1D2 cell cultures were mocktreated or exposed to IFN-β (1,000 U/mL). After 24 h, the cells were harvestedand the relative amounts of Sp100 and Daxx were quantified, as described inMaterials and Methods. mRNA levels of Sp100 and Daxx were normalized to18S RNA, and their relative value in untreated group were set as 1. Two-tailedP values were calculated using standard t test.

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PML. To determine the role of ICP0 in defining the size of theaggregates early in infection, a monolayer culture of parentalHEp-2 or 1D2 cells was exposed to 10 pfu HSV-1 per cell. After2 h, the cultures were fixed and reacted with monoclonal anti-body to PML and polyclonal antibody to ICP0. Typical imagesshown in Fig. 4 A–C indicate that the ND10 bodies containingboth PML and ICP0 are uniform in size in HEp-2 cells. Incontrast, ICP0 aggregates formed in infected 1D2 cells variedconsiderably in size (Fig. 4 D–F).

The Role of PML in the Degradation of Sp100. Studies publishedelsewhere have shown that ICP0 is an E3 ligase that, in conjunctionwith UbcH5a ubiquitin-conjugating enzyme, degrades PML andSp100 (9–11). Mutations in the ring finger located in exon 2 of ICP0abolish the ligase activity (11). The question posed here is whetherthe absence of PML hinders the degradation of Sp100. We reporttwo series of experiments. In the first, parental HEp-2 cells or 1D2cells grown on four-well slides were infected with 10 pfu HSV-1(F)or a ring finger (RF) mutant that is bereft of ubiquitin ligase ac-tivity. At 3 h after exposure to the virus, the cells were fixed andreacted with antibody to ICP0 and to Sp100, as described earlier.The results may be summarized as follows: First, Fig. 5 A–C

shows two 1D2 cells. The cell labeled A contains numerous ag-gregates of Sp100 differing in size and relatively few aggregates ofICP0. As shown in Fig. 5C, only a few of the aggregates of ICP0(green) colocalized with the aggregates of Sp100 (red). Moreover,as apparent in Fig. 5C, the colocalized structures did not fullymerge. In the cell labeled B, there are few prominent Sp100 bodies.The one prominent Sp100 body colocalized but did not merge withthe ICP0 (green) aggregates.Second, Fig. 5 D–F shows two parental HEp-2 cells. The cell

labeled C shows no signs of being infected. Thus, Sp100 aggregatesare prominent, and no ICP0 can be seen. In contrast, there waslittle Sp100 and very prominent ICP0 aggregates in small uniformstructures consistent with the size of ND10 bodies in the cell la-beled D. The data are consistent with the hypothesis that Sp100had been degraded.Finally, in parental (PML+/+) HEp-2 cells infected with RF mu-

tant (Fig. 5 J–L), ICP0 and Sp100 aggregated in small nuclearstructures relatively uniform in size indistinguishable from classicalND10 bodies. In contrast, in 1D2 cells, there was no evidence oflarge-scale colocalization of ICP0 and Sp100. Although Sp100

formed aggregates differing in size, ICP0 appeared to diffuse in thenucleus (Fig. 5 G–I).The results presented here suggest that in PML−/− cells, the

colocalization of ICP0 and Sp100 is a slow process and may notresult in a total mixing of the two proteins. The paucity of Sp100 inFig. 5C (cell labeled “B”) suggests Sp100 was degraded, possibly bymassive accumulation of ICP0.To quantify the pattern of decay of SP100 in the two cell lines,

replicate cultures of HEp-2 or ID2 cells were transfected withFLAG-tagged SP100, as described in Materials and Methods. After48 h, the cells were exposed to 10 pfu HSV-1(F) per cell. Culturesharvested at time of exposure to the virus (Mock) or at 3, 5, 7, or10 h after infection were solubilized, subjected to electrophoresis indenaturing gels, and probed with antibody to Sp100 and β-actin.Fig. 6A shows the immunoblots of Sp100 and β-actin. Fig. 6Bshows the results of a density scan normalized with respect toSp100 present in mock-treated cells. The results show that under

Fig. 3. Cellular localization of Sp100 and Daxx in HEp-2 and 1D2 cells. At 70% confluency, the medium of parental HEp-2 or 1D2 cells grown on four-well slideswas replaced with fresh medium alone or medium supplemented with IFN-β (1,000 U/mL). After 24 h, the cells were fixed and reacted with polyclonal antibody toSp100 (red) and either PML (green, A) or Daxx (green, B). (Magnification: 100×.)

Fig. 4. ICP0 formed aggregates in PML−/− cells. HEp-2 (A–C) and 1D2 cells (D–F)grown on four-well slides were treated with IFN-β, as earlier, and then exposed to10 pfu HSV-(F) per cell. At 2 h after infection, the cells were fixed and reactedwithpolyclonal antibody to ICP0 (red) and monoclonal antibody to PML (green).(Magnification: 100×.)

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conditions used in these studies, the initiation of decay of Sp100in 1D2 cells was delayed by at least 3 h, in contrast to the ap-parent initiation of decay of Sp100, which extrapolates to a timeat or shortly after exposure of the cells to the virus.

The Replication of Wild-Type Virus Is Impaired in 1D2 Cells Infected at LowRatios of Virus per Cell. The degradation of PML mediated by ICP0E3 ligase early after infection suggests that PML is inimical to thereplication of HSV-1. To test this hypothesis, we exposed replicatecultures of parentalHEp-2 or 1D2 cells to either 0.1 or 5 pfuHSV-1(F)per cell. The cells were harvested at intervals between 2 and 48 h inthe case of cultures exposed to 5 pfu per cell, or between 2 and 72 hin the case of cultures exposed to 0.1 pfu virus per cell. As shown inFig. 7B, the virus yields from cultures exposed to 5 pfu per cell werevirtually identical. In contrast, the yields of virus obtained 48 and 72 hafter infection of 1D2 cells with 0.1 pfu per cell were∼100-fold lowerthan those obtained in parental HEp-2 cells exposed to the sameratio of virus per cell (Fig. 7A). The results suggest that PML mayplay both a supportive and inimical role in HSV-1 replication.

The Accumulation of ICP0 Is Impaired in 1D2 Cells Infected at LowRatios of Virus per Cell. One hypothesis that could explain the re-duced accumulation of virus in 1D2 cells at low multiplicities ofinfection is a reduction in the synthesis of ICP0. To test this hy-pothesis, replicate cultures of HEp-2 or 1D2 cells were infectedwith either 10 or 0.1 pfu virus per cell. At intervals after infectionshown in Fig. 8, the cells were harvested, solubilized, subjected toelectrophoresis in denaturing gels, transferred to a nitrocellulosesheet, and probed with antibody to ICP0 and β-actin. The resultsshown in Fig. 8 indicate that the rates of accumulations of ICP0 inHEp-2 or 1D2 cells exposed to 10 pfu per cell were approximatelyequal. In contrast, the accumulation of ICP0 in 1D2 cells laggedsignificantly behind the accumulation of ICP0 observed in HEp-2cells. The significance of the results rests on two considerations:Foremost, the synthesis and accumulation of ICP0 is dependent onthe presence of PML, even though ICP0 ultimately degrades PML,and second, the delay in the degradation of Sp100, as shown in Fig.5, is not a result of the lack of ICP0, as in those experiments, thecells were exposed to 10 pfu per cell.

Effect of IFN-β on the Replication of HSV-1(F) in Parental HEp-2 and in1D2 Cells. Earlier studies have shown that IFN-β is more effective insuppressing HSV-1 replication in primary murine fibroblast culturesthan in sibling cells in which PML was knocked out. The questionarose whether IFN-β had a similar effect in human cells, and inparticular, in the parental HEp-2 and in PML−/− 1D2 clone cellsused in the studies reported here. In the experiments reported,replicate cultures of the two cell lines were exposed to 0.1 pfu HSV-1per cell and then either incubated for 24 h without further treatmentor maintained in medium containing IFN. The results are shown inFig. 9. As expected from the results in Fig. 7, the yields of untreated1D2 cells were lower than those obtained in parental HEp-2 cells. InIFN-β–treated cells, the yield of virus at 15 h was reduced 100-foldcompared with those obtained in untreated infected cells. In con-trast, IFN-β decreased the yield of virus in 1D2 cells 10-fold com-pared with yields obtained in infected untreated 1D2 cells. Inessence, the results obtained in a human cell line paralleled thoseobtained in murine cells.

DiscussionThe function, and ultimately the degradation, of PML in HSV-1-infected cells has been the subject of numerous studies. The pre-ponderance of evidence supports the hypothesis that PML is inimicalto virus replication. Thus, the accumulation of PML is enhanced

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Fig. 5. (A–L) ICP0 and Sp100 aggregated with Daxx do not colocalize efficientlyin 1D2 cells infected with wild-type or RF mutant viruses. Cells grown on four-well slide cultures and treated as earlier were exposed to 10 pfu wild-type or RFmutant virus per cell. At 3 h after infection, the cultures were fixed and reactedwith monoclonal antibody to ICP0 and polyclonal antibody to Sp100. The imagesshown were acquired using a Zeiss confocal microscope. (Magnification: 100×.)

Fig. 6. Degradation of Sp100 by HSV-1(F) infection is delayed in PML−/− 1D2cells. (A) HEp-2 and 1D2 cells were transfected with a plasmid-encoding Flag-tagged Sp100. At 48 h after transfection, the cells were exposed to 10 pfu wild-type virus per cell. At 0 (mock), 3, 5, 7, or 10 h after infection, the cells wereharvested, solubilized, subjected to electrophoresis in denatured gels, andreacted with antibody to Sp100 and β-actin. (B) The relative amounts of Flag-tagged Sp100 were quantified and normalized to β-actin, and the amounts ofFlag-tagged Sp100 in mock-treated cells (0 h after infection) with the aid ofImage J. The degradation rates of FLAG-Sp100 were calculated as described inMaterials and Methods.

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after exposure of cells to IFN-β (26). In addition, as reinforced in thisstudy, the antiviral effects of IFN-β are drastically reduced in theabsence of PML (27). Finally, one of the first and most prominentactions of ICP0, a product made immediately after infection, is todegrade PML and Sp100, another key component of ND10 bodies.The enhancement of synthesis of PML and Sp100 by IFN-β and thetargeting of these two components of ND10 bodies for degradationmay be selective. Thus, Daxx, an ND10 constituent that colocalizeswith Sp100, is neither enhanced by IFN-β nor degraded by ICP0.PML appears to be an effector of INF-β, and one expectation of ourstudies was that the absence of PML would be beneficial to HSVreplication. This is, in fact, not the case. Our findings are as follows:In PML−/− cells, the typical ND10 bodies were replaced with bodiescontaining at least Daxx and Sp100. These bodies vary in size, but onaverage, are larger than the typical ND10 bodies present in parentalPML+/+ cells. In PML−/− cells, ICP0 formed aggregates that in somecells partially colocalized but did not merge with the SP100/Daxxbodies. Consistent with the evidence that colocalization of ICP0 andSp100/Daxx bodies was not a common event, Sp100 was degraded,but only after a delay of several hours. The delay in the degradationof Sp100 was not a result of a lack of ICP0 inasmuch as, underconditions tested, cells infected with 10 pfu virus per cell, the two celllines accumulated equivalent amounts of ICP0. We conclude thatsome constituent of ND10 bodies aggregate in the absence of PML.PML appears to confer typical morphology to ND10 bodies, and theaggregation of ICP0 near ND10 bodies ultimately leads to the deg-radation of SP100. In addition, at high multiplicities of infection, thepattern of accumulation of infectious virus in PML−/− cells is notsignificantly different from that in parental PML+/+. In contrast, incells infected with 0.1 pfu per cell, the yields were 100-fold lower thanin PML+/+ cells exposed to the same ratio of virus per cell. Equallyunexpected, the amounts of ICP0 that accumulated in PML−/− cells

exposed to 0.1 pfu per cell was significantly lower than that producedin PML+/+ cells exposed to the same ratio of virus per cell.The data presented in this report do not support the current

consensus on the events taking place in the infected cells immedi-ately after entry of viral DNA into the nucleus. Thus, the currentconsensus is that on entry of viral DNA into the nucleus, numerouscellular proteins attempt to transcriptionally silence viral DNA (1, 7,8, 35–37). Concurrently, VP16, a key viral transcriptional factor,recruits cellular protein to transcribe and enable the synthesis of αproteins (38–41). In addition, concurrently or sequentially, a ND10body assembles at viral DNA (7, 8), and one α protein, ICP0, in-teracts with PML, gains entry, and diffuses throughout the ND10body (12). Finally, ICP0 associated with the ND10 body degradesPML and Sp100 (9, 10, 42). According to this sequence of events,the synthesis of α proteins is independent of the interaction of virioncomponents with PML. The data presented here suggest that op-timal accumulation of viral α proteins, and ultimately the accumu-lation of viral proteins, is enhanced by PML.An intriguing question is why the parental HEp-2 cells infected at

high or very low ratios of pfu per cell produced identical yields,whereas 1D2 cells infected at low ratios of virus per cell producedsignificantly lower yields than cells infected at high ratios of virus percell. Several nonexclusive mechanisms may be responsible. Specifi-cally, in principle, in cultures infected at low ratios of virus per cell,the final yield is the sum of virus produced during multiple cycles ofinfection, replication, and spread to uninfected cells. The inherentassumption has been that the yield of virus from the first cycle isidentical to that of the last cycle. This concept has been challengedby the observation that cells export along with infectious particlesexosomes containing micro-RNAs that may affect the synthesis ofvirus gene products and inimical cellular innate immune factors (e.g.,STING) (43, 44). It is conceivable that the negative effect of exo-somes is much higher on late cycles than on earlier ones.In addition, the effect on viral replication in cells contracting

concurrently both virus and the contents of exosomes bearing an-tiviral factors may be quite different from that taking place in cellsin which entry of antiviral factors born by exosomes precedes virusentry by several hours. The delay in the degradation of SP100 in

Fig. 7. Growth of HSV-1(F) in PML−/− 1D2 cells is impaired in PML−/− cells ex-posed to low ratios of virus per cell. Replicate cultures of HEp-2 or 1D2 cellswere exposed to 5 or 0.1 pfu virus per cell. At times shown, the cells wereharvested and virus yields were titered in Vero cell.

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Fig. 8. (A and B) Accumulation of ICP0 is reduced in PML−/−1D2 cells exposedto low ratios of virus per cell. Replicate cultures of HEp-2 or 1D2 cells wereexposed to 10 or 0.1 pfu virus per cell. Cells were harvested at indicated pointsand were reacted with antibody to ICP0 and β-actin.

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cells exposed to 10 pfu per cell suggests that assembly of virusfactories required for optimal accumulation of gene products incells infected at lower multiplicities may be even more delayed.Last, two highly significant studies showed that in infected cells,

the number of virus factories or assemblons initiated by singlegenomes is less than 10, irrespective of the multiplicity of infection(45, 46). Implicit in this finding is the notion that either a criticalcomponent necessary for the assembly of virus factories is limitedin supply, or that there is a gradual accumulation of innate immunefactors that curtail the formation of virus factories once a criticalnumber is formed. A convenient but unsupported hypothesis thatcould explain our data is that in the absence of PML, the resistanceto formation of virus factories increases.Irrespective of whether the hypotheses are ultimately borne out

by further studies, the results of this report indicate that HSV-1evolved the means to take advantage of an inimical cellular de-fense mechanism to degrade it, and at the same time use it to gainaccess to a nuclear domain essential for efficient replication.

Materials and MethodsCells and Viruses. The sources and maintenance of HEP-2 and Vero cells werereported elsewhere (47). HSV-1(F) is the prototype strain used in ourlaboratory (48). RF-HSV (F) was a recombinant virus containing ring-fingerdomain deletion of both copies of ICP0 protein described elsewhere (12).All viruses were amplified in HEp-2 cells. Plaque assays were performed inVero cells.

RNA Isolation and Real-Time PCR. RNA extraction and assays were done aspreviously described (49). mRNA level of Sp100 and Daxx were measured bygene-specific primers: Sp100 forward primer, 5′-GCTCAGGACCCCAGATTGTAC-3′,and reverse primer, 5′-CTAATCTTCTTTACCTGACCC-3′, and Daxx forward primer,5′-GACGGACATTTCCTCTTCCA-3′, and reverse primer, 5′-CGCCTCCATTGAAGG-AAGTA-3′.

Transfection Plus Superinfection. Parental HEp-2 and PML−/− clone 1D2 cellswere grown to 70–90% confluency in T150 flasks. Each flask was transfectedwith 18 μg of a plasmid DNA containing a CMV promoter-driven FLAG-taggedSp100 ORF, using Lipofectamine LTX with Plus Reagent (Life Technologies,15338100), according to manufacturer’s instructions. At 24 h after transfection,the cells contained in T150 flasks were split into T25 flasks. At 24 h afterseeding, the mock-transfected and FLAG-tagged Sp100-transfected cells weremock infected or exposed to 10 pfu HSV-1(F) per cell. The cells were harvestedand processed as described in Results.

Generation of PML−/− Cell Line Using CRISPR.ACRISPR kit targeting the first exonof PML genewas obtained fromOrigene (cat. no. KN200700). Parental HEp-2 cellswere cotransfected with a gRNA vector plasmid targeting sequences 5′-CTCGGA-GATCGGGCGGGTGC-3′ (150th–169th bp in exon 1; 4th–10th amino acid from thestart codon) or 5′-GGCCTTCAGAGGGGGTCTCG-3′ (218th–237th bp in exon1; 26th–32nd amino acid from the start codon), and a donor vector containing a selectioncassette expressing GFP–puromycin was inserted into the targeted site in humanPML gene. Selected cells (GFP+ and puromycin resistant) were serially diluted toform single cell-derived colonies. The colonies were further screened for expressionof PML by immunoblotting and immunofluorescence.

Immunoblot Analyses. At indicated times, the cells were lysed, heat denatured,electrophoretically separated in denaturing gels, transferred to polyvinylidenedifluoride membranes, and probed with the appropriate primary antibody, aspreviously described (49). The antibodies includedmonoclonal anti-PML (SantaCruz, sc-966) (50), rabbit polyclonal anti-PML (Abcam, 79466), mouse mono-clonal anti–β-actin (Thermo Fisher, MA5-15739), rabbit polyclonal anti-ICP0(Lab stock), and mouse monoclonal anti-FLAG (Sigma, F1804). The membraneswere then incubated with alkaline phosphatase-conjugated goat anti-mouseor goat anti-rabbit secondary antibody (Sigma) and visualized with BCIP/NBTWestern Blotting Detection Reagent (GE Healthcare).

ImageJ Quantification and Statistics. Image J software was used to quantifyband intensity. FLAG-Sp100 levels were first normalized to β-actin. Degradationrates of FLAG-Sp100 were calculated by normalization to Sp100 in mock-treated cells. Error bars were calculated from the results of three independentquantifications of band intensities. Two-tailed P values were calculated usingstandard t test. To compare ICP0 expression level, the band intensity of ICP0wasfirst normalized to β-actin. For high multiplicity of infection, ICP0 expressionlevel of all points examined was then normalized to that in HEp-2 at 24 hpostinfection; for low multiplicity of infection, ICP0 expression level of all pointsexamined was then normalized to that in HEp-2 at 48 h postinfection.

Immunofluorescence Staining and Confocal Microscopy. Cells in four-welledslides were fixed and permeabilized, blocked, and then incubated with primaryantibodies, as previously described (47). Those included antibodies anti-PML,as described earlier, rabbit anti-Sp100 (Abcam, ab43151); mouse anti-Daxx(Abcam, ab9091); and rabbit anti-ICP0 or mouse anti-ICP0, described elsewhere(47), at 4 °C overnight, rinsed three times with blocking solution, and subjectedto reaction with fluorescein isothiocyanate-conjugated goat anti-mouse (Invi-trogen) or/and Texas Red-conjugated goat anti-rabbit (Sigma) secondary anti-bodies. Images were taken at 100× magnification with a Zeiss confocalmicroscopy.

ACKNOWLEDGMENTS. We thank Lindsay Smith for invaluable assistance; andGuoying Zhou, Rozanne Sandri-Goldin, and David Bloom for careful reading ofthe article and useful comments and suggestions. These studies were aided bya grant from the Joseph Regenstein Foundation.

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P<0.05108

107

106

PFU

109

HEp-2 1D2

105

P=0.0018

Fig. 9. The antiviral effects of IFN-β are diminished in cells in PML−/− cells.HEp-2 or PML−/− 1D2 cells were pretreated with IFN-β at 1,000 U/mL for 24 hand then exposed to 0.1 pfu virus per cell. The cells were harvested at 24 hafter infection. Virus yields were titered in Vero cells. Antiviral effects of IFN-βtreatment in HEp-2 or PML−/− 1D2 cells were represented by viral titer re-duction posttreatment. P value was calculated by comparison of antiviral ef-fects of IFN-β in HEp-2 and that in PML−/− 1D2 cells.

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