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.     

Herpes Simplex Virus Type 1 Regulatory Protein ICP0 Aids Infection in Cells with a Preinduced Interferon Response but Does Not Impede Interferon-Induced Gene Induction

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.The innate immune response mediated through the interferon (IFN) pathway is an important component of antiviral defense mediated by individual cells and whole organisms (10, 28). In turn, many viruses express proteins that counteract the effects of the IFN response (28). In the case of herpes simplex virus type 1 (HSV-1), highly defective HSV-1 mutants activate expression of IFN-stimulated genes (ISGs) through a mechanism that is independent of IFN itself but dependent on IFN regulatory factor 3 (IRF-3) (2, 3, 19, 23, 26). HSV-1 mutants that do not express the immediate-early (IE) regulatory protein ICP0 are more sensitive than the wild-type (wt) virus to IFN pretreatment of cultured cells (13, 20), and ICP0-null mutant HSV-1 is much more pathogenic in mice unable to respond to IFN (12, 15). Furthermore, a number of experimental systems have presented evidence suggesting that a specific function of ICP0 is to interfere with IFN and/or IRF-3-dependent IFN responses (3, 16-18, 21). However, we have reported recently that the replication defect of ICP0-null mutant HSV-1 is not complemented in cultured cells lacking either STAT1 or IRF-3 (9), which raises the question of whether the relative sensitivity of ICP0-null mutant HSV-1 to an IFN-induced antiviral state results from the absence of a specific effect of ICP0 on IFN pathways or is, rather, an indirect consequence of the disabled virus being intrinsically less able to replicate in cells expressing ISGs (9).The investigation of these complex issues is difficult because sensitivity to IFN is highly dependent on multiplicity of infection (MOI) (9) and cell type (20). Therefore, we sought to develop a system in which the specific effects of ICP0 could be examined in the absence of HSV-1 infection and which avoids potential complications arising from the use of viral vectors or plasmid transfection technologies. In an accompanying paper, we describe the construction of a cell line that expresses ICP0 at physiological levels in an inducible manner (7). The cells allow 100% complementation of plaque formation by ICP0-null mutant HSV-1, and induction of ICP0 expression induces efficient reactivation of gene expression from quiescent HSV-1 genomes (7). We have used these cells to investigate whether, by itself, ICP0 is able to impede induction of ISGs in response to IFN (through the normal STAT1 signaling pathway) or to interfere with IRF-3-dependent activation of ISGs induced by double-stranded RNA, the archetypal pathogen-associated molecular pattern (PAMP). We found that preexpression of ICP0 had no deleterious effect on either pathway. On the other hand, preexpression of ICP0 decreased (but did not eliminate) the sensitivity of HSV-1 to an IFN-induced antiviral state. We discuss the relationship between ICP0 and intrinsic and innate cellular defenses to HSV-1 infection.     

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A)⁺ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral infection. ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2, 3, 5, 10, 13, 21, 34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20, 21). ICP27 also interacts with the export adaptor proteins Aly/REF (2, 3, 23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5′ end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11, 36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16, 17, 27) by binding RNA and interacting with TAP/NXF1 (14, 16, 18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of newly transcribed RNA during infection (11). Thus, these SR proteins also contribute to the efficiency of viral RNA export. However, the overexpression of SRp20 was unable to rescue the defect in RNA export during infection with an ICP27 mutant that cannot bind RNA (11), suggesting that ICP27 is the major HSV-1 RNA export protein that links viral RNA to TAP/NXF1.ICP27 was shown previously to bind RNA through an RGG box motif located at amino acids 138 to 152 within the 512-amino-acid protein (28, 34). Using electrophoretic mobility shift assays (EMSAs), we showed that the N-terminal portion of ICP27 from amino acids 1 to 160 bound specifically to viral oligonucleotides that are GC rich and that are flexible and relatively unstructured (5). Here we report the importance of three arginine residues within the RGG box for ICP27 binding to GC-rich sequences in vitro and for viral RNA export during infection. We also performed nuclear magnetic resonance (NMR) structural analysis of the N-terminal portion of ICP27 for both the wild-type protein and an ICP27 mutant in which three arginines were replaced with lysines. The NMR data showed that the N-terminal portion of ICP27 is relatively unstructured but compact, and NMR analysis in the presence of oligonucleotide substrates to which the N-terminal portion of ICP27 binds did not show any discernible alterations in this highly flexible structure, nor did the arginine-to-lysine substitutions.     

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.