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Intrinsic immunity is an aspect of antiviral defense that operates through diverse mechanisms at the intracellular level through a wide range of constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the repression of viral gene expression during the very early stages of infection, a process that is normally overcome by viral tegument and/or immediate-early proteins. Thus, the balance between cellular repressors and virus-counteracting proteins determines whether or not a cell becomes productively infected. One aspect of intrinsic resistance to herpes simplex virus 1 (HSV-1) is conferred by components of promyelocytic leukemia nuclear bodies (PML NBs), which respond to infection by accumulating at sites that are closely associated with the incoming parental HSV-1 genomes. Other cellular proteins, including IFI16, which has been implicated in sensing pathogen DNA and initiating signaling pathways that lead to an interferon response, also respond to viral genomes in this manner. Here, studies of the dynamics of the response of PML NB components and IFI16 to invading HSV-1 genomes demonstrated that this response is extremely rapid, occurring within the first hour after addition of the virus, and that human Daxx (hDaxx) and IFI16 respond more rapidly than PML. In the absence of HSV-1 regulatory protein ICP0, which counteracts the recruitment process, the newly formed, viral-genome-induced PML NB-like foci can fuse with existing PML NBs. These data are consistent with a model involving viral genome sequestration into such structures, thereby contributing to the low probability of initiation of lytic infection in the absence of ICP0. [ABSTRACT FROM AUTHOR]

Covalent linkage to members of the small ubiquitin-like (SUMO) family of proteins is an important mechanism by which the functions of many cellular proteins are regulated. Sumoylation has roles in the control of protein stability, activity and localization, and is involved in the regulation of transcription, gene expression, chromatin structure, nuclear transport and RNA metabolism. Sumoylation is also linked, both positively and negatively, with the replication of many different viruses both in terms of modification of viral proteins and modulation of sumoylated cellular proteins that influence the efficiency of infection. One prominent example of the latter is the widespread reduction in the levels of cellular sumoylated species induced by herpes simplex virus type 1 (HSV-1) ubiquitin ligase ICP0. This activity correlates with relief from intrinsic immunity antiviral defence mechanisms. Previous work has shown that ICP0 is selective in substrate choice, with some sumoylated proteins such the promyelocytic leukemia protein PML being extremely sensitive, while RanGAP is completely resistant. Here we present a comprehensive proteomic analysis of changes in the cellular SUMO2 proteome during HSV-1 infection. Amongst the 877 potentially sumoylated species detected, we identified 124 whose abundance was decreased by a factor of 3 or more by the virus, several of which were validated by western blot and expression analysis. We found many previously undescribed substrates of ICP0 whose degradation occurs by a range of mechanisms, influenced or not by sumoylation and/or the SUMO2 interaction motif within ICP0. Many of these proteins are known or are predicted to be involved in the regulation of transcription, chromatin assembly or modification. These results present novel insights into mechanisms and host cell proteins that might influence the efficiency of HSV-1 infection. [ABSTRACT FROM AUTHOR]

Herpes simplex virus 1 (HSV-1) regulatory protein ICP0 stimulates efficient infection via its E3 ubiquitin ligase activity that causes degradation of several cellular proteins, some of which are sumoylated. Chicken adenovirus Gam1 protein also interferes with the sumoylation pathway, and both proteins disrupt promyelocytic leukemia protein (PML) nuclear bodies (NBs). We report that Gam1 increases the infection efficiency of ICP0-null mutant HSV-1 by approximately 100-fold, thus strengthening the hypothesis that PML NB- and sumoylation-related mechanisms are important factors in the control of HSV-1 infection. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 immediate-early protein ICP0 is an E3 ubiquitin ligase of the RING finger class that degrades several cellular proteins during infection. This activity is essential for its functions in stimulating efficient lytic infection and productive reactivation from latency. ICP0 targets a number of proteins that are modified by the small ubiquitin-like SUMO family of proteins, and it includes a number of short sequences that are related to SUMO interaction motifs (SIMs). Therefore, ICP0 has characteristics that are related to those of cellular SUMO-targeted ubiquitin ligase enzymes. Here, we analyze the impact of mutation of a number of SIM-like sequences (SLSs) within ICP0 on HSV-1 replication and gene expression and their requirement for ICP0-mediated degradation of both sumoylated and unmodified promyelocytic leukemia (PML) and other sumoylated cellular proteins. One SLS in the central portion of the ICP0 sequence (SLS4) was found to be absolutely required for targeting cellular sumoylated species in general and sumoylated forms of PML other than those of PML isoform I. Mutation of a group of SLSs in the C-terminal quarter of ICP0 also reduced ICP0-mediated degradation of sumoylated PML in a cooperative manner. Although mutation of individual SLSs caused only modest decreases in viral replication, combined mutation of SLS4 with SLS sequences in the C-terminal quarter of the protein reduced plaque formation efficiency by up to two orders of magnitude. These results provide further evidence that the biological activities of ICP0 are connected with host cell sumoylation events. IMPORTANCE Herpes simplex virus type 1 protein ICP0 plays important roles in regulating the initial stages of lytic infection and productive reactivation from latency. ICP0 mediates its effects through inducing the degradation of cellular proteins that have repressive effects on viral gene expression. An increasing number of cellular proteins are known to be sensitive to ICP0-mediated degradation; therefore, it is important to understand how ICP0 selects its substrates for degradation. This study identifies sequence motifs within ICP0 that are involved in targeting cellular proteins that are modified by the SUMO family of ubiquitin-like proteins and describes how mutation of combinations of these motifs causes a 100-fold defect in viral infectivity. [ABSTRACT FROM AUTHOR]

Intrinsic antiviral resistance is a branch of antiviral defence that involves constitutively expressed cellular proteins that act within individual infected cells. In recent years it has been discovered that components of cellular nuclear structures known as ND10 or PML nuclear bodies contribute to intrinsic resistance against a variety of viruses, notably of the herpesvirus family. Several ND10 components are rapidly recruited to sites that are closely associated with herpes simplex virus type 1 (HSV-1) genomes during the earliest stages of infection, and this property correlates with the efficiency of ND10 mediated restriction of HSV-1 replication. Similar but distinct recruitment of certain DNA damage response proteins also occurs during infection. These recruitment events are inhibited in a normal wild type HSV-1 infection by the viral regulatory protein ICP0. HSV-1 mutants that do not express ICP0 are highly susceptible to repression through intrinsic resistance factors, but they replicate more efficiently in cells depleted of certain ND10 proteins or in which ND10 component recruitment is inefficient. This article presents the background to this recruitment phenomenon and summaries how it is conveniently studied by fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Abstract: Laser scanning confocal microscopy is a powerful technique that can be applied to study the localisation and behaviour of proteins and nucleic acids in many experimental situations. It is a particularly useful technique for the study of virus infections because of the changes that occur in the distribution and amounts of both viral and cellular proteins as infection develops. These changes reflect key stages and important regulatory events that govern the efficiency of infection. Using herpes simplex virus type 1 infected cells as an experimental model, this article provides guidance for users new to confocal microscopy on basic principles and techniques. The emphasis is on recognising, diagnosing and avoiding potential artifacts, and the workflow of the production of high quality, technically correct images. [Copyright &y& Elsevier]

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMOmodified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection. [ABSTRACT FROM AUTHOR]

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway. [ABSTRACT FROM AUTHOR]

It has been proposed that the cellular corepressor protein CoREST is involved in repressing herpes simplex virus type 1 (HSV-1) infection in the absence of the viral regulatory protein ICP0. This proposal predicts that depletion of CoREST should improve the plaque-forming efficiency and replication of ICP0 null mutant virus. To test this hypothesis, human HepaRG cells that were highly depleted of CoREST were isolated using RNA interference technology. Depletion of CoREST had no effect on the replication of ICP0 null mutant HSV-1, demonstrating that CoREST does not play an influential role in regulating HSV-1 infection in this cell type. [ABSTRACT FROM AUTHOR]

Immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is an E3 ubiquitin ligase of the RING finger class that is required for efficient lytic infection and reactivation from latency. Other alphaherpesviruses also express ICP0-related RING finger proteins, but these have limited homology outside the core RING domain. Existing evidence indicates that ICP0 family members have similar properties, but there has been no systematic comparison of the biochemical activities and biological functions of these proteins. Here, we describe an inducible cell line system that allows expression of the ICP0-related proteins of bovine herpes virus type 1 (BHV-1), equine herpesvirus type 1 (EHV-1), pseudorabies virus (PRV), and varicella-zoster virus (VZV) and their subsequent functional analysis. We report that the RING domains of all the proteins have E3 ubiquitin ligase activity in vitro. The BHV-1, EHV-1, and PRV proteins complement ICP0-null mutant HSV-1 plaque formation and induce derepression of quiescent HSV-1 genomes to levels similar to those achieved by ICP0 itself. VICP0, the ICP0 expressed by VZV, was found to be extremely unstable, which limited its analysis in this system. We compared the abilities of the ICP0-related proteins to disrupt ND10, to induce degradation of PML and Sp100, to affect key components of the interferon signaling pathway, and to interfere with induction of interferon-stimulated genes. We found that the property that correlated most closely with their biological activities was the ability to preclude the recruitment of cellular ND10 proteins to sites closely associated with incoming HSV-1 genomes and early replication compartments. [ABSTRACT FROM AUTHOR]

Infections with DNA viruses commonly result in the association of viral genomes and replication compartments with cellular nuclear substructures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. While there is evidence that viral genomes can associate with preexisting ND10, we demonstrate in this study by live-cell microscopy that structures resembling ND10 form de novo and in association with viral genome complexes during the initial stages of herpes simplex virus type 1 (HSV-1) infection. Consistent with previous studies, we found that the major ND10 proteins PML, Sp100, and hDaxx are exchanged very rapidly between ND10 foci and the surrounding nucleoplasm in live cells. The dynamic nature of the individual protein molecule components of ND10 provides a mechanism by which ND10 proteins can be recruited to novel sites during virus infection. These observations explain why the genomes and replication compartments of DNA viruses that replicate in the cell nucleus are so commonly found in association with ND10. These findings are discussed with reference to the nature, location, and potential number of HSV-1 prereplication compartments and to the dynamic aspects of HSV-1 genomes and viral products during the early stages of lytic infection. [ABSTRACT FROM AUTHOR]

ND10 structures are disrupted during herpes simplex virus type 1 (HSV-1) infection by viral regulatory protein ICP0. The significance of this effect remains controversial, partly because of a report that high-level expression of the major ND10 promyelocytic leukemia (PML) protein precludes ND10 disruption yet does not inhibit HSV-1 infection. Here we demonstrate dramatic reorganization of ND10 during HSV-1 infection by live-cell microscopy, even in the presence of overexpressed PML. [ABSTRACT FROM AUTHOR]

Previous reports have suggested that herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stabilizes cyclins D1 and D3 during infection by inducing the degradation of cdc34, the E2-conjugating enzyme that is responsible for regulating the stability of these cyclins. Since ICP0 has complex effects on the progress of viral infection that vary greatly with cell type and viral dose, it can be difficult to distinguish between direct effects caused by ICP0 itself and indirect effects caused by the rate of the progression of infection in the absence of ICP0 at the chosen multiplicity of infection. This report describes the fates of cdc34 and cyclins D1 and D3 during HSV-1 infection under conditions that ensured that viral infection and gene expression were proceeding at equivalent rates in the presence and absence of ICP0. It was confirmed that both D-type cyclins were unstable during HSV-1 infection of a variety of cell types, but no effect on cdc34 was observed, even when high levels of ICP0 were expressed. Furthermore, there was no evidence that ICP0 protected either cyclin D1 or cyclin D3 from degradation. Reconstruction of the conditions of the experiments in the previous studies, using the stated cell type and multiplicities of infection, indicated that the original results could be explained by differences in the rate of progression of infection rather than by the presence or absence of ICP0. The data presented in this report are incompatible with the hypothesis that ICP0 induces the degradation of cdc34 and thereby stabilizes cyclins D1 and D3 during HSV-1 infection. [ABSTRACT FROM AUTHOR]

The major oncoprotein p53 regulates several cellular antiproliferation pathways that can be triggered in response to a variety of cellular stresses, including viral infection. The stabilization of p53 is a key factor in the ability of cells to initiate an efficient transcriptional response after cellular stress. Here we present data demonstrating that herpes simplex virus type 1 (HSV-1) infection of HFFF-2 cells, a low-passage-number nontransformed human primary cell line, results in the stabilization of p53. This process required viral immediate-early gene expression but occurred independently of the viral regulatory protein ICP0 and viral DNA replication. No specific viral protein could be identified as being solely responsible for the effect, which appears to be a cellular response to developing HSV-1 infections. HSV-1 infection also induced the phosphorylation of p53 at residues Ser15 and Ser20, which have previously been implicated in its stabilization in response to DNA damage. However, an HSV-1 infection of ATM-/- cells, which lack a kinase implicated in these phosphorylation events, did not lead to the phosphorylation of p53 at these residues, but nonetheless p53 was stabilized. We also show that the wild-type p53 expressed by osteosarcoma U2OS cells can be stabilized in response to DNA damage induced by UV irradiation, but not in response to HSV-1 infection. These data suggest that multiple cellular mechanisms are initiated to stabilize p53 during an HSV-1 infection. These mechanisms occur independently of ICP0 and its ability to sequester USP7 and may differ from those initiated in response to DNA damage. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) has an intricate association with cellular nuclear structures known as ND10 or promyelocytic leukemia protein (PML) nuclear bodies. Parental viral genomes initially become juxtaposed to ND10, and then viral replication compartments develop from the ND10-associated genomes. Viral immediate-early (IE) regulatory protein ICP0 colocalizes with ND10 and then induces the degradation of critical ND10 component protein PML and therefore the release and dispersal of other ND10 proteins. The IE transcriptional regulatory protein ICP4 also forms foci at early times of infection, many of which are juxtaposed to ND10 and later develop into replication compartments, indicating that at least some of the initial ICP4 foci contain parental viral genomes. Here we report that the ICP4 foci also contain ICP27 and that their formation occurs extremely rapidly at locations just inside the nuclear envelope. By examining developing plaques or thinly seeded cells infected at high multiplicity, we found evidence to suggest that at least some of the ND10-viral nucleoprotein complex association could be attributed to de novo formation of ND10-1ike structures in response to incoming viral genomes. The ICP4 complexes associated efficiently with ND10 in cells infected with an ICP0-null mutant virus at high but not at low multiplicity, and the degree of association was reduced by the proteasome inhibitor MG132. Therefore, the interaction between viral nucleoprotein complexes and ND10 is in part due to a dynamic response by the cell. This response is modulated by functional ICP0, and cells that are productively or nonproductively infected in the absence of functional ICP0 can be distinguished by the relative locations of ICP4 foci and ND10 proteins. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein ICP0 is required for efficient progression of infected cells into productive lytic infection, especially in low-multiplicity infections of limitedpassage human fibroblasts. We have used single-cell-based assays that allow detailed analysis of the ICP0-null phenotype in low-multiplicity infections of restrictive cell types. The major conclusions are as follows: (i) there is a threshold input multiplicity above which the mutant virus replicates normally; (ii) individual cells infected below the threshold multiplicity have a high probability of establishing a nonproductive infection; (iii) such nonproductively infected cells have a high probability of expressing IE products at 6 h postinfection; (iv) even at 24 h postinfection, IE protein-positive nonproductively infected human fibroblast cells exceed the number of cells that lead to plaque formation by up to 2 orders of magnitude; (v) expression of individual IE proteins in a proportion of the nonproductively infected cells is incompletely coordinated; (vi) the nonproductive cells can also express early gene products at low frequencies and in a stochastic manner; and (vii) significant numbers of human fibroblast cells infected at low multiplicity by an ICP0-deficient virus are lost through cell death. We propose that in the absence of ICP0 expression, HSV-1 infected human fibroblasts can undergo a great variety of fates, including quiescence, stalled infection at a variety of different stages, cell death, and, for a minor population, initiation of formation of a plaque. [ABSTRACT FROM AUTHOR]

Demonstrates that the herpes simplex virus type 1 regulatory protein ICP0 is a genuine RING finger ubiquitin E3 ligase that can interact with and mediate with the ubiquitination of the major oncoprotein p53 both in vitro and in vivo. Requirement in the ubiquitination of p43 for ICP0 to have an intact RING finger domain and occurs independently of its ability to bind to the ubiquitin-specific protease SUP7.

Examination of cells at the early stages of herpes simplex virus type 1 infection revealed that the viral immediate-early protein Vmw110 (also known as ICP0) formed discrete punctate accumulations associated with centromeres in both mitotic and interphase cells. The RING finger domain of Vmw110 (but not the C-terminal region) was essential for its localization at centromeres, thus distinguishing the Vmw110 sequences required for centromere association from those required for its localization at other discrete nuclear structures known as ND10, promyelocytic leukaemia (PML) bodies or PODs. We have shown recently that Vmw110 can induce the proteasome-dependent loss of several cellular proteins, including a number of probable SUMO-1-conjugated isoforms of PML, and this results in the disruption of ND10. In this study, we found some striking similarities between the interactions of Vmw110 with ND10 and centromeres. Specifically, centromeric protein CENP-C was lost from centromeres during virus infection in a Vmw110- and proteasome-dependent manner, causing substantial ultrastructural changes in the kinetochore. In consequence, dividing cells either became stalled in mitosis or underwent an unusual cytokinesis resulting in daughter cells with many micronuclei. These results emphasize the importance of CENP-C for mitotic progression and suggest that Vmw110 may be interfering with biochemical mechanisms which are relevant to both centromeres and ND10. [ABSTRACT FROM AUTHOR]

The ability of herpes simplex virus type 1 (HSV-1) to attain a latent state in sensory neurones and reactivate periodically is crucial for its biological and clinical properties. The active transcription of the entire 152 kb viral genome during lytic replication contrasts with the latent state, which is characterized by the production of a single set of nuclear-retained transcripts. Reactivation of latent genomes to re-initiate the lytic cycle therefore involves a profound change in viral transcriptional activity, but the mechanisms by which this fundamentally important process occurs are yet to be well understood. In this report we show that the stimulation of the onset of viral lytic infection mediated by the viral immediate-early (IE) protein Vmw110 is strikingly inhibited by inactivation of the ubiquitin—proteasome pathway. Similarly, the Vmw110-dependent reactivation of quiescent viral genomes in cultured cells is also dependent on proteasome activity. These results constitute the first demonstration that the transcriptional activity of a viral genome can be regulated by protein stability control pathways. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 immediate-early protein Vmw110 is a non-specific activator of gene expression and is required for efficient initiation of the viral lytic cycle. Since Vmw110-deficient viruses reactivate inefficiently in mouse latency models it has been suggested that Vmw110 plays a role in the balance between the latent and lytic states of the virus. The mechanisms by which Vmw110 achieves these functions are poorly understood. Vmw110 migrates to discrete nuclear structures (ND10) which contain the cellular PML protein, and in consequence PML and other constituent proteins are dispersed. In addition, Vmw110 binds to a cellular protein of ∼135 kDa, and its interactions with the 135 kDa protein and ND10 contribute to its ability to stimulate gene expression and viral lytic growth. In this report we identify the 135 kDa protein as a novel member of the ubiquitin-specific protease family. The protease is distributed in the nucleus in a micropunctate pattern with a limited number of larger discrete foci, some of which co-localize with PML in ND10. At early times of virus infection, the presence of Vmw110 increases the proportion of ND10 which contain the ubiquitin-specific protease. These results identify a novel, transitory component of ND10 and implicate a previously uncharacterized ubiquitindependent pathway in the control of viral gene expression. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) immediate-early gene product ICP0 activates lytic infection and relieves cell-mediated repression of viral gene expression. This repression is conferred by preexisting cellular proteins and is commonly referred to as intrinsic antiviral resistance or intrinsic defense. PML and Sp100, two core components of nuclear substructures known as ND10 or PML nuclear bodies, contribute to intrinsic resistance, but it is clear that other proteins must also be involved. We have tested the hypothesis that additional ND10 factors, particularly those that are involved in chromatin remodeling, may have roles in intrinsic resistance against HSV-1 infection. The two ND10 component proteins investigated in this report are ATRX and hDaxx, which are known to interact with each other and comprise components of a repressive chromatin-remodeling complex. We generated stable cell lines in which endogenous ATRX or hDaxx expression is severely suppressed by RNA interference. We found increases in both gene expression and plaque formation induced by ICP0-null mutant HSV-1 in both ATRX- and hDaxx-depleted cells. Reconstitution of wild-type hDaxx expression reversed the effects of hDaxx depletion, but reconstitution with a mutant form of hDaxx unable to interact with ATRX did not. Our results suggest that ATRX and hDaxx act as a complex that contributes to intrinsic antiviral resistance to HSV-1 infection, which is counteracted by ICP0. [ABSTRACT FROM AUTHOR]

Several independent lines of evidence indicate that interferon-mediated innate responses are involved in controlling herpes simplex virus type 1 (HSV-1) infection and that the viral immediate-early regulatory protein ICP0 augments HSV-1 replication in interferon-treated cells. However, this is a complex situation in which the experimental outcome is determined by the choice of multiplicity of infection and cell type and by whether cultured cells or animal models are used. It is now known that neither STAT1 nor interferon regulatory factor 3 (IRF-3) play essential roles in the replication defect of ICP0-null mutant HSV-1 in cultured cells. This study set out to investigate the specific role of ICP0 in HSV-1 resistance to the interferon defense. We have used a cell line in which ICP0 expression can be induced at levels similar to those during the early stages of a normal infection to determine whether ICP0 by itself can interfere with interferon or IRF-3-dependent signaling and whether ICP0 enables the virus to circumvent the effects of interferon-stimulated genes (ISGs). We found that the presence of ICP0 was unable to compromise ISG induction by either interferon or double-stranded RNA. On the other hand, ICP0 preexpression reduced but did not eliminate the inhibitory effects of ISGs on HSV-1 infection, with the extent of the relief being highly dependent on multiplicity of infection. The results are discussed in terms of the relationships between ICP0 and intrinsic and innate antiviral resistance mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) genomes become associated with structures related to cellular nuclear substructures known as ND10 or promyelocytic leukemia nuclear bodies during the early stages of lytic infection. This paper describes the relationship between HSV-1 genomes and ND10 in human fibroblasts that maintain the viral genomes in a quiescent state. We report that quiescent HSV-1 genomes detected by fluorescence in situ hybridization (FISH) are associated with enlarged ND10-like structures, frequently such that the FISH-defined viral foci are apparently enveloped within a sphere of PML and other ND10 proteins. The number of FISH viral foci in each quiescently infected cell is concordant with the input multiplicity of infection, with each structure containing no more than a small number of viral genomes. A proportion of the enlarged ND10-like foci in quiescently infected cells contain accumulations of the heterochromatin protein HP1 but not other common markers of heterochromatin such as histone H3 di- or trimethylated on lysine residue 9. Many of the virally induced enlarged ND10-like structures also contain concentrations of conjugated ubiquitin. Quiescent infections can be established in cells that are highly depleted for PML. However, during the initial stages of establishment of a quiescent infection in such cells, other ND10 proteins (Sp100, hDaxx, and ATRX) are recruited into virally induced foci that are likely to be associated with HSV-1 genomes. These observations illustrate that the intimate connections between HSV-1 genomes and ND10 that occur during lytic infection also extend to quiescent infections. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus (HCMV) tegument protein pp71, encoded by gene UL82, stimulates viral immediate-early (IE) transcription. pp71 interacts with the cellular protein hDaxx at nuclear domain 10 (ND10) sites, resulting in the reversal of hDaxx-mediated repression of viral transcription. We demonstrate that pp71 displaces an hDaxx-binding protein, ATRX, from ND10 prior to any detectable effects on hDaxx itself and that this event contributes to the role of pp71 in alleviating repression. Introduction of pp71 into cells by transfection, infection with a pp71-expressing herpes simplex virus type 1 vector, or by generation of transformed cell lines promoted the rapid relocation of ATRX from ND10 to the nucleoplasm without alteration of hDaxx levels or localization. A pp71 mutant protein unable to interact with hDaxx did not affect the intranuclear distribution of ATRX. Infection with HCMV at a high multiplicity of infection resulted in rapid displacement of ATRX from ND10, the effect being observed maximally by 2 h after adsorption, whereas infection with the UL82-null HCMV mutant ADsubUL82 did not affect ATRX localization even at 7 h postinfection. Cell lines depleted of ATRX by transduction with shRNA-expressing lentiviruses supported increased IE gene expression and virus replication after infection with ADsubUL82, demonstrating that ATRX has a role in repressing IE transcription. The results show that ATRX, in addition to hDaxx, is a component of cellular intrinsic defenses that limit HCMV IE transcription and that displacement of ATRX from ND10 by pp71 is important for the efficient initiation of viral gene expression. [ABSTRACT FROM AUTHOR]

During the early stages of herpes simplex virus type 1 (HSV-1) infection, viral immediate-early regulatory protein ICP0 localizes to and disrupts cellular nuclear structures known as PML nuclear bodies or ND10. These activities correlate with the functions of ICP0 in stimulating lytic infection and reactivating quiescent HSV-1. The disruption of ND10 occurs because ICP0 induces the loss of the SUMO-1-modified forms of PML and the subsequent proteasome-mediated degradation of the PML protein. The functions of ICP0 are largely dependent on the integrity of its zinc-binding RING finger domain. Many RING finger proteins have been found to act as ubiquitin E3 ligase enzymes, stimulating the production of conjugated polyubiquitin chains in the presence of ubiquitin, the ubiquitin-activating enzyme E1, and the appropriate E2 ubiquitin-conjugating enzyme. Substrate proteins that become polyubiquitinated are then subject to degradation by proteasomes. We have previously shown that purified full-length ICP0 acts as an efficient E3 ligase in vitro, producing highmolecular-weight polyubiquitin chains in a RING finger-dependent but substrate-independent manner. In this paper we report on investigations into the factors governing the degradation of PML induced by ICP0 in a variety of in vivo and in vitro assays. We found that ICP0 expression increases the levels of ubiquitinated PML in transfected cells. However, ICP0 does not interact with or directly ubiquitinate either unmodified PML or SUMO-1-modified PML in vitro, suggesting either that additional factors are required for the ICP0-mediated ubiquitination of PML in vivo or that PML degradation is an indirect consequence of some other activity of ICP0 at ND10. Using a transfection-based approach and a family of deletion and point mutations of PML, we found that efficient ICP0-induced PML degradation requires sequences within the C-terminal part of PML and iysine residue 160, one of the principal targets for SUMO-1... [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 stimulates lytic infection and the reactivation of quiescent viral genomes. These roles of ICP0 require its RING finger E3 ubiquitin ligase domain, which induces the degradation of several cellular proteins, including components of promyelocytic leukemia nuclear bodies and centromeres. ICP0 also interacts very strongly with the cellular ubiquitin-specific protease USP7 (also known as HAUSP). We have shown previously that ICP0 induces its own ubiquitination and degradation in a RING finger-dependent manner, and that its interaction with USP7 regulates this process. In the course of these studies we found and report here that ICP0 also targets USP7 for ubiquitination and proteasome-dependent degradation. The reciprocal activities of the two proteins reveal an intriguing situation that poses the question of the balance of the two processes during productive HSV-1 infection. Based on a thorough analysis of the properties of an HSV-1 mutant virus that expresses forms of ICP0 that are unable to bind to USP7, we conclude that USP7-mediated stabilization of ICP0 is dominant over ICP0-induced degradation of USP7 during productive HSV-1 infection. We propose that the biological significance of the ICP0-USP7 interaction may be most pronounced in natural infection situations, in which limited amounts of ICP0 are expressed. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 immediate-early regulatory protein ICP0 stimulates lytic infection and reactivation from latency, processes that require the ubiquitin E3 ligase activity mediated by the RING finger domain in the N-terminal portion of the protein. ICP0 stimulates the production of polyubiquitin chains by the ubiquitin-conjugating enzymes UbcH5a and UbcH6 in vitro, and in infected and transfected cells it induces the proteasome-dependent degradation of a number of cellular proteins including PML, the major constituent protein of PML nuclear bodies. However, ICP0 binds strongly to the cellular ubiquitin-specific protease USP7, a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors. The region of ICP0 that is required for its interaction with USP7 has been mapped, and mutations in this domain reduce the functionality of ICP0. These findings pose the question: why does ICP0 include domains that are associated with the potentially antagonistic functions of ubiquitin conjugation and deconjugation? Here we report that although neither protein affected the intrinsic activities of the other in vitro, TJSP7 protected ICP0 from autoubiquitination in vitro, and their interaction can greatly increase the stability of ICP0 in vivo. These results demonstrate that RING finger-mediated autoubiquitination of ICP0 is biologically relevant and can be regulated by interaction with USP7. This principle may extend to a number of cellular RING finger E3 ubiquitin ligase proteins that have analogous interactions with ubiquitin-specific cleavage enzymes. [ABSTRACT FROM AUTHOR]

To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The viral ubiquitin ligase ICP0 is required for efficient initiation of herpes simplex virus 1 (HSV-1) lytic infection and productive reactivation of viral genomes from latency. ICP0 has been shown to target a number of specific cellular proteins for proteasome-dependent degradation during lytic infection, including the promyelocytic leukemia protein (PML) and its small ubiquitin-like modified (SUMO) isoforms. We have shown previously that ICP0 can catalyze the formation of unanchored polyubiquitin chains and mediate the ubiquitination of specific substrate proteins in vitro in the presence of two E2 ubiquitin-conjugating enzymes, namely, UBE2D1 (UbcH5a) and UBE2E1 (UbcH6), in a RING finger-dependent manner. Using homology modeling in conjunction with site-directed mutagenesis, we identify specific residues required for the interaction between the RING finger domain of ICP0 and UBE2D1, and we report that point mutations at these residues compromise the ability of ICP0 to induce the colocalization of conjugated ubiquitin and the degradation of PML and its SUMO-modified isoforms. Furthermore, we show that RING finger mutants that are unable to interact with UBE2D1 fail not only to complement the plaque-forming defect of an ICP0-null mutant virus but also to mediate the derepression of quiescent HSV-1 genomes in cell culture. These data demonstrate that the ability of ICP0 to interact with cellular E2 ubiquitin-conjugating enzymes is fundamentally important for its biological functions during HSV-1 infection [ABSTRACT FROM AUTHOR]

Expression of the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 in transfected cells reactivates rep gene expression from integrated adeno-associated virus (AAV) type 2 genomes via a mechanism that requires both its RING finger and USP7 interaction domains. In this study, we found that the rep reactivation defect of USP7-binding-negative ICP0 mutants can be overcome by further deletion of sequences in the C-terminal domain of ICP0, indicating that binding of USP7 to ICP0 is not directly required. Unlike the case in transfected cells, only the RING finger domain of ICP0 was essential for rep gene reactivation during HSV-1 infection. However, mutants unable to bind to USP7 activate HSV-1 gene expression and reactivate rep gene expression with reduced efficiencies. These results further elucidate the role of ICP0 as a helper factor for AAV replication and illustrate that care is required when extrapolating from the properties of ICP0 in transfection assays to events occurring during HSV-1 infection. [ABSTRACT FROM AUTHOR]