Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects

Herpes simplex virus (HSV) 1 disaggregates the nuclear domain 10 (ND10) nuclear structures and disperses its organizing promyelocytic leukemia protein (PML). An earlier report showed that ectopic overexpression of PML precludes the disaggregation of ND10 but has no effect on viral replication. PML has been reported to mediate the effects of interferon (IFN) and viral mutants lacking ICP0 (Delta alpha 0 mutants). To test the hypothesis that HSV disaggregates ND10 structures and disperses PML to preclude IFN-mediated antiviral effects, we tested the accumulation of viral proteins and virus yields from murine PML(+/+) and PML(-/-) cells mock treated or exposed to IFN-alpha, IFN-gamma, or both and infected with the wild-type or Delta alpha 0 mutant virus. We report the following results. (i) The levels of growth of wild-type and mutant viruses and of accumulation of viral proteins were not significantly different in untreated PML(+/+) and PML(-/-) cells. (ii) Major effects of IFN-alpha and -gamma were observed in PML(+/+) cells infected with the Delta alpha 0 mutant virus, and more minor effects were observed in cells infected with the wild-type virus. The effects of the IFNs on either wild-type or the mutant virus in PML(-/-) cells were minimal. (iii) The mixture of IFN-alpha and -gamma was more effective than either IFN alone, but again, the effect was more drastic in PML(+/+) cells than in PML(-/-) cells. We concluded that the anti-HSV state induced by exogenous IFN is mediated by PML and that the virus targets the ND10 structures and disseminates PML in order to preclude the establishment of the antiviral state induced by IFNs.     

Overexpression of promyelocytic leukemia protein precludes the dispersal of ND10 structures and has no effect on accumulation of infectious herpes simplex virus 1 or its proteins

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the M(r) 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of alpha, beta, or gamma groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the alpha 0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.     

Role of cyclin D3 in the biology of herpes simplex virus 1 ICPO

Earlier reports from this laboratory have shown that the promiscuous transactivator infected-cell protein 0 (ICP0) binds and stabilizes cyclin D3, that the binding site maps to aspartic acid 199 (D199), and that replacement of D199 with alanine abolishes binding and reduces the capacity of the mutant virus to replicate in quiescent cells or to cause mortality in mice infected by a peripheral site. The objective of this report was to investigate the role of cyclin D3 in the biology of ICP0. We report the following results. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stabilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cytoplasm between 3 and 9 h. after infection, ICPO containing the D199A substitution remains quantitatively in the nucleus. (iv) To examine the interaction of ICP0 with cyclin D3, we used a previously described mutant carrying a wild-type ICP0 but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substituted ICP0. Early in infection with R7801, ICP0 colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICP0 was translocated to the cytoplasm whereas cyclin D3 remained in the nucleus. The translocation of ICP0 to the cytoplasm was accelerated in cells expressing cyclin D3 compared with that of ICP0 expressed by wild-type virus. In contrast, ICP0 carrying the D199A substitution remained in the nucleus and did not colocalize with cyclin D3. These studies suggest the following conclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in consequence, an activated cdk4. The metabolic events occurring at or near that structure and involving cyclin D3 cause the translocation of ICP0 to the cytoplasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function responsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced.     

PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0

Promyelocytic leukemia (PML) nuclear bodies (also known as ND10) are nuclear substructures that contain several proteins, including PML itself, Sp100, and hDaxx. PML has been implicated in many cellular processes, and ND10 are frequently associated with the replicating genomes of DNA viruses. During herpes simplex virus type 1 (HSV-1) infection, the viral regulatory protein ICP0 localizes to ND10 and induces the degradation of PML, thereby disrupting ND10 and dispersing their constituent proteins. ICP0-null mutant viruses are defective in PML degradation and ND10 disruption, and concomitantly they initiate productive infection very inefficiently. Although these data are consistent with a repressive role for PML and/or ND10 during HSV-1 infection, evidence in support of this hypothesis has been inconclusive. By use of short interfering RNA technology, we demonstrate that depletion of PML increases both gene expression and plaque formation by an ICP0-negative HSV-1 mutant, while having no effect on wild-type HSV-1. We conclude that PML contributes to a cellular antiviral repression mechanism that is countered by the activity of ICP0.     

Herpes simplex virus 1 gene expression is accelerated by inhibitors of histone deacetylases in rabbit skin cells infected with a mutant carrying a cDNA copy of the infected-cell protein no. 0

An earlier report showed that the expression of viral genes by a herpes simplex virus 1 mutant [HSV-1(vCPc0)] in which the wild-type, spliced gene encoding infected-cell protein no. 0 (ICP0) was replaced by a cDNA copy is dependent on both the cell type and multiplicity of infection. At low multiplicities of infection, viral gene expression in rabbit skin cells was delayed by many hours, although ultimately virus yield was comparable to that of the wild-type virus. This defect was rescued by replacement of the cDNA copy with the wild-type gene. To test the hypothesis that the delay reflected a dysfunction of ICP0 in altering the structure of host protein-viral DNA complexes, we examined the state of histone deacetylases (HDACs) (HDAC1, HDAC2, and HDAC3). We report the following. (i) HDAC1 and HDAC2, but not HDAC3, were modified in infected cells. The modification was mediated by the viral protein kinase U(S)3 and occurred between 3 and 6 h after infection with wild-type virus but was delayed in rabbit skin cells infected with HSV-1(vCPc0) mutant, concordant with a delay in the expression of viral genes. (ii) Pretreatment of rabbit skin cells with inhibitors of HDAC activity (e.g., sodium butyrate, Helminthosporium carbonum toxin, or trichostatin A) accelerated the expression of HSV-1(vCPc0) but not that of wild-type virus. We conclude the following. (i) In the interval in which HSV-1(vCPc0) DNA is silent, its DNA is in chromatin-like structures amenable to modification by inhibitors of histone deacetylases. (ii) Expression of wild-type virus genes in these cells precluded the formation of DNA-protein structures that would be affected by either the HDACs or their inhibitors. (iii) Since the defect in HSV-1(vCPc0) maps to ICP0, the results suggest that this protein initiates the process of divestiture of viral DNA from tight chromatin structures but could be replaced by other viral proteins in cells infected with a large number of virions.     

Human neuron-committed teratocarcinoma NT2 cell line has abnormal ND10 structures and is poorly infected by herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.     

The differential requirement for cyclin-dependent kinase activities distinguishes two functions of herpes simplex virus type 1 ICP0

Herpes simplex virus type 1 (HSV-1) ICP0 directs the degradation of cellular proteins associated with nuclear structures called ND10, a function thought to be closely associated with its broad transactivating activity. Roscovitine (Rosco), an inhibitor of cyclin-dependent kinases (cdks), inhibits the replication of HSV-1, HSV-2, human cytomegalovirus, varicella-zoster virus, and human immunodeficiency virus type 1 by inhibiting specific steps or activities of viral regulatory proteins, indicating the broad and pleiotropic effects that cdks have on the replication of these viruses. We previously demonstrated that Rosco inhibits the transactivating activity of ICP0. In the present study, we asked whether Rosco also affects the ability of ICP0 to direct the degradation of ND10-associated proteins. For this purpose, WI-38 cells treated with cycloheximide (CHX) were mock infected or infected with wild-type HSV-1 or an ICP0(-) mutant (7134). After release from the CHX block, the infections were allowed to proceed for 2 h in the presence or absence of Rosco at a concentration known to inhibit ICP0's transactivating activity. The cells were then examined for the presence of ICP0 and selected ND10-associated antigens (promyelocytic leukemia antigen [PML], sp100, hDaxx, and NDP55) by immunofluorescence. Staining for the ND10-associated antigens was detected in 90% of 7134- and mock-infected cells stained positive for all ND10-associated antigens in the presence or absence of Rosco. Similar results were obtained with a non-ND10-associated antigen, DNA-PK(cs), a known target of ICP0-directed degradation. The results of the PML and DNA-PK(cs) immunofluorescence studies correlated with a decrease in the levels of these proteins as determined by Western blotting. Thus, the differential requirement for Rosco-sensitive cdk activities distinguishes ICP0's ability to direct the dispersal or degradation of cellular proteins from its transactivating activity.     

Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0''s RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.Alphaherpesviruses encode orthologs of the herpes simplex virus (HSV) α gene product ICP0. ICP0 is a nuclear phosphoprotein that behaves as a promiscuous activator of viral and cellular genes (7, 11, 28, 29). ICP0 also functions as an E3 ubiquitin ligase to target several host proteins for proteasomal degradation (4, 10, 11, 16, 26). Through this activity, ICP0 promotes degradation of components of nuclear domain 10 (ND10) bodies, including the promyelocytic leukemia protein (PML) and Sp100. These proteins are implicated in silencing of herpesvirus genomes (9, 10, 22, 34). Therefore, ICP0-mediated degradation of ND10 components may disrupt silencing of HSV genes to enable efficient gene expression. This hypothesis provides a plausible mechanistic explanation of how ICP0 induces gene activation.Introduction of DNA encoding the ICP0 orthologs from HSV, bovine herpesvirus, equine herpesvirus, and varicella-zoster virus (VZV) can also affect nuclear structures and proteins (27). In addition, and more specific to this report, ORF61p, the VZV ortholog, activates viral promoters and enhances infectivity of viral DNA like ICP0, the prototype for this gene family (24, 25). However, we have previously demonstrated two key biological differences between the HSV and VZV orthologs. We first showed that unlike ICP0, ORF61p is unable to complement depletion of BAG3, a host cochaperone protein. As a result, VZV is affected by silencing of BAG3 (15), whereas growth of HSV is altered only when ICP0 is not expressed (17). Furthermore, we have shown that while both proteins target components of ND10s, expression of ICP0 results in degradation of both PML and Sp100, whereas ORF61p specifically reduces Sp100 levels (16). These findings suggest that these proteins have evolved separately to provide different functions for virus replication.Virus mutants lacking the ICP0 gene have an increased particle-to-PFU ratio, a substantially lower yield, and decreased levels of α gene expression, in a multiplicity-of-infection (MOI)- and cell-type-dependent manner (2, 4, 8, 33). These mutants are also defective at degrading ND10 components (23). Depletion of PML and Sp100 accelerates virus gene expression and increases plaquing efficiency of HSV ICP0-defective viruses but has no effect on wild-type virus, suggesting that PML and Sp100 are components of an intrinsic anti-HSV defense mechanism that is counteracted by ICP0''s E3 ligase activity (9, 10). Interestingly, ICP0 null viruses are also hypersensitive to interferon (IFN) (26), a property that was suggested to be mediated via PML (3).To directly compare the activities of the two orthologs, we constructed an HSV mutant virus that expresses ORF61p in place of ICP0. The resulting chimeric virus only partially rescues the ICP0 null phenotype. Our studies emphasize the biological differences between ICP0 and ORF61p and shed light on the requirements for PML and Sp100 during infection.     

The Replication Defect of ICP0-Null Mutant Herpes Simplex Virus 1 Can Be Largely Complemented by the Combined Activities of Human Cytomegalovirus Proteins IE1 and pp71

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0.     

ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.     

The Viral Ubiquitin Ligase ICP0 Is neither Sufficient nor Necessary for Degradation of the Cellular DNA Sensor IFI16 during Herpes Simplex Virus 1 Infection

The cellular protein IFI16 colocalizes with the herpes simplex virus 1 (HSV-1) ubiquitin ligase ICP0 at early times of infection and is degraded as infection progresses. Here, we report that the factors governing the degradation of IFI16 and its colocalization with ICP0 are distinct from those of promyelocytic leukemia protein (PML), a well-characterized ICP0 substrate. Unlike PML, IFI16 colocalization with ICP0 was dependent on the ICP0 RING finger and did not occur when proteasome activity was inhibited. Expression of ICP0 in the absence of infection did not destabilize IFI16, the degradation occurred efficiently in the absence of ICP0 if infection was progressing efficiently, and IFI16 was relatively stable in wild-type (wt) HSV-1-infected U2OS cells. Therefore, IFI16 stability appears to be regulated by cellular factors in response to active HSV-1 infection rather than directly by ICP0. Because IFI16 is a DNA sensor that becomes associated with viral genomes during the early stages of infection, we investigated its role in the recruitment of PML nuclear body (PML NB) components to viral genomes. Recruitment of PML and hDaxx was less efficient in a proportion of IFI16-depleted cells, and this correlated with improved replication efficiency of ICP0-null mutant HSV-1. Because the absence of interferon regulatory factor 3 (IRF3) does not increase the plaque formation efficiency of ICP0-null mutant HSV-1, we speculate that IFI16 contributes to cell-mediated restriction of HSV-1 in a manner that is separable from its roles in IRF3-mediated interferon induction, but that may be linked to the PML NB response to viral infection.     

STAT-1- and IRF-3-dependent pathways are not essential for repression of ICP0-null mutant herpes simplex virus type 1 in human fibroblasts

Efficient herpes simplex virus type 1 (HSV-1) infection of human fibroblasts (HFs) is highly dependent on the viral immediate-early regulatory protein ICP0 unless the infection is conducted at a high multiplicity. ICP0-null mutant HSV-1 exhibits a plaque-forming defect of up to 3 orders of magnitude in HFs, whereas in many other cell types, this defect varies between 10- and 30-fold. The reasons for the high ICP0 requirement for HSV-1 infection in HFs have not been established definitively. Previous studies using other cell types suggested that ICP0-null mutant HSV-1 is hypersensitive to interferon and that this sensitivity is dependent on the cellular promyelocytic leukemia (PML) protein. To investigate the roles of two important aspects of interferon signaling in the phenotype of ICP0-null mutant HSV-1, we isolated HFs depleted of STAT-1 or interferon regulatory factor 3 (IRF-3). Surprisingly, plaque formation by the mutant virus was not improved in either cell type. We found that the sensitivity to interferon pretreatment of both ICP0-null mutant and wild-type (wt) HSV-1 was highly dependent on the multiplicity of infection. At a low multiplicity in virus yield experiments, both viruses were extremely susceptible to interferon pretreatment of HFs, but the sensitivity of the wild type but not the mutant could be overcome at higher multiplicities. We found that both wt and ICP0-null mutant HSV-1 remained sensitive to interferon in PML-depleted HFs albeit to an apparently lesser extent than in control cells. The data imply that the substantial reduction in ICP0-null HSV-1 infectivity at a low multiplicity in HFs does not occur through the activities of STAT-1- and IRF-3-dependent pathways and cannot be explained solely by enhanced sensitivity to interferon. We suggest that antiviral activities induced by interferon may be separable from and additive to those resulting from PML-related intrinsic resistance mechanisms.     

Herpes Simplex Virus 1 Ubiquitin Ligase ICP0 Interacts with PML Isoform I and Induces Its SUMO-Independent Degradation

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction.     

Analysis of the Functions of Herpes Simplex Virus Type 1 Regulatory Protein ICP0 That Are Critical for Lytic Infection and Derepression of Quiescent Viral Genomes

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 is important for stimulating the initiation of the lytic cycle and efficient reactivation of latent or quiescent infection. Extensive investigation has suggested several potential functions for ICP0, including interference in the interferon response, disruption of functions connected with PML nuclear bodies (ND10), and inhibition of cellular histone deacetylase (HDAC) activity through an interaction with the HDAC-1 binding partner CoREST. Analysis of the significance of these potential functions and whether they are direct or indirect effects of ICP0 is complicated because HSV-1 mutants expressing mutant forms of ICP0 infect cells with widely differing efficiencies. On the other hand, transfection approaches for ICP0 expression do not allow studies of whole cell populations because of their limited efficiency. To overcome these problems, we have established a cell line in which ICP0 expression can be induced at levels pertaining during the early stages of HSV-1 infection in virtually all cells in the culture. Such cells enable 100% complementation of ICP0-null mutant HSV-1. Using cells expressing the wild type and a variety of mutant forms of ICP0, we have used this system to analyze the role of defined domains of the protein in stimulating lytic infection and derepression from quiescence. Activity in these core functions correlated well the ability of ICP0 to disrupt ND10 and inhibit the recruitment of ND10 proteins to sites closely associated with viral genomes at the onset of infection, whereas the CoREST binding region was neither sufficient nor necessary for ICP0 function in lytic and reactivating infections.Herpes simplex virus type 1 (HSV-1) is an important human pathogen that infects the majority of the population at an early age and then establishes a life-long latent infection in sensory neurones. Periodic reactivation of latent virus causes episodes of active disease characterized by epithelial lesions at the site of the original primary infection. As with all herpesviruses, the ability of HSV-1 to establish and reactivate from latency is key to its clinical importance and evolutionary success. Therefore, the molecular mechanisms that regulate these processes have been the subject of intensive research (reviewed in reference 15). HSV-1 immediate-early (IE) protein ICP0 is required for efficient reactivation from latency in both mouse models and cultured cell systems of quiescent infection (15). ICP0 is also required to stimulate lytic infection by enhancing the probability that a cell receiving a viral genome will engage in productive infection (reviewed in references 19, 20 and 42). Therefore, a full understanding of the biology of HSV-1 infection requires a definition of the functions and mode of action of ICP0.The basic phenotype of ICP0-null mutant HSV-1 is a low probability of plaque formation, particularly in human diploid fibroblasts, that causes a high particle-to-PFU ratio (reference 20 and references therein). Biochemically, ICP0 is an E3 ubiquitin ligase of the RING finger class (4) that induces the degradation of several cellular proteins, including the promyelocytic leukemia (PML) protein (23), centromere proteins including CENP-C (54, 55), and the catalytic subunit of DNA-protein kinase (53, 72). Among the consequences of these activities are the disruption of PML nuclear bodies (herein termed nuclear domain 10 [ND10]) (24, 58) and centromeres (54). ICP0 has also been reported to interact with histone deacetylase enzymes (HDACs) (56) and the CoREST repressor protein, thereby disrupting the CoREST/HDAC-1 complex (37, 39). Evidence has also been presented that expression of ICP0 correlates with increased acetylation of histones on viral chromatin (12). ICP0-null mutant viruses replicate less efficiently than the wild type (wt) in cells pretreated with interferon (IFN) (44, 66), and there is evidence that ICP0 is able to impede an IFN-independent induction of IFN-stimulated genes that arises after infection with defective HSV-1 mutants (16, 59, 60, 65, 67, 76). As a further complication, ICP0-null mutant HSV-1 replicates more efficiently in cells that have been highly stressed by a variety of treatments (5, 6, 79).On the basis of this evidence, several not necessarily mutually exclusive hypotheses have been put forward to explain the biological effects of ICP0. These include (i) that ICP0 counteracts an intrinsic cellular resistance mechanism that involves PML and other components of ND10, (ii) that ICP0 overcomes the innate cellular antiviral defense based on the IFN pathway, and (iii) that ICP0 counteracts the establishment of a repressed chromatin structure on the viral genome by interfering with histone deacetylation. The aim of this paper is to investigate some of these issues using a novel inducible expression system. The question of the effects of ICP0 on IFN pathways is considered in the companion paper (28).The brief and by no means exhaustive summary of the functions and activities attributed to ICP0, presented above, illustrates that the understanding of ICP0 is a difficult issue. It is further complicated by the difficulty of working with ICP0-null mutant viruses under tightly controlled conditions. This arises because the defect varies greatly between different cell types, is highly dependent on the multiplicity of infection (MOI), and varies in a nonlinear manner with respect to virus dose (reference 20 and references therein). Furthermore, use of ICP0 mutant viruses in cultured cell models of reactivation of quiescent HSV-1 is complicated by competition between the resident quiescent viral genome targeted for reactivation and the genomes of the superinfecting virus used to induce the reactivation (75). Therefore, it is very difficult to establish infections with wt and ICP0 mutant viruses that are truly comparable in a way that allows clear distinctions between the direct effects of ICP0 and indirect effects that are due either to expression of other viral proteins that are expressed more efficiently in the presence of ICP0 or to less specific consequences of an active infection and subsequent effects on the cell. Here, we describe a system that enables expression of ICP0 in an inducible manner at levels similar to those at the early stages of infection in almost all cells in a population. We have used this system to study wt and mutant forms of ICP0 in assays of lytic infection and derepression of quiescent viral genomes in a cultured cell model of latency. We discuss the results in terms of the requirements of specific regions of the ICP0 protein for stimulating lytic infection and derepression of quiescent genomes, the potential biological significance of ND10 disruption, recruitment of ND10 components to the sites of HSV-1 genomes at the outset of virus infection, and the interaction of ICP0 with CoREST.