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Journal of Virology, May 2009, p. 4963-4977, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02593-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Roger D. Everett,* Marie-Laure Parsy, and Anne Orr

MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, Scotland, United Kingdom

Received 16 December 2008/ Accepted 23 February 2009


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ABSTRACT
 
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.


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INTRODUCTION
 
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.


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MATERIALS AND METHODS
 
Viruses and cells. HSV-1 strain 17+ was the wt strain used, from which was derived the ICP0-null mutant dl1403. Viruses in1863 and dl1403/CMVlacZ are derivatives of the above that contain the lacZ gene under the control of the human cytomegalovirus (HCMV) promoter/enhancer inserted into the tk gene (kindly provided by Chris Preston). HSV-1 mutant virus in1374 contains the tsK temperature-sensitive lesion in ICP4, a deletion of the ICP0 gene, and a mutation within VP16 that inactivates its ability to stimulate IE gene expression (78). HSV-1 mutants with specific deletion mutations in ICP0 (FXE, D8, D12, D13, D14, and E52X) have been described previously (23, 61). All viruses were grown in BHK (baby hamster kidney) cells and titrated in U2OS cells, in which ICP0 is not required for efficient replication of HSV-1. Virus in1374 was propagated at the permissive temperature of 31°C and grown in the presence of 2.5 mM hexamethylbisacetamide (78). U2OS, HEK-293T, and human fibroblast (HF) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. BHK cells were grown in Glasgow modified Eagle's medium supplemented with 10% newborn calf serum and 10% tryptose phosphate broth. HepaRG hepatocyte cells (36) were grown in William's medium E supplemented with 10% fetal bovine serum Gold (PAA Laboratories Ltd.), 2 mM glutamine, 5 µg/ml insulin, and 0.5 µM hydrocortisone. All cell growth media were supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. Lentivirus-transduced cells were maintained with continuous antibiotic selection, as appropriate.

Plasmids. Lentivirus vector plasmids expressing proteins from RNA polymerase II (Pol II) promoters were constructed by removing the RNA Pol III and short hairpin RNA sequences from pLKO.shPML1 (based on pLKO.1puro) (27) and replacing them with a fragment containing either the complete HCMV IE promoter enhancer region or a truncated version of this promoter (DCMV) with its 5' limit at the NdeI site in the HCMV IE enhancer. The lentivirus vector backbone included either a puromycin resistance gene (pLKO series plasmids) or a neomycin/G418 resistance gene (pLKOneo series). For tetracycline-inducible lentiviruses, a tandem TetO operator sequence was inserted at the SacI site located 10 bp 3' of the DCMV promoter TATA box. Plasmids were constructed so that selected cDNA open reading frames could be inserted downstream of the Pol II promoter. Plasmid pLKOneo.CMV.EGFPnlsTetR includes the complete HCMV promoter/enhancer upstream of a fusion protein expressing enhanced green fluorescent protein (EGFP) linked to a nuclear localization signal (NLS) and the tetracycline repressor (TetR). The EGFPnlsTetR fusion protein has been described previously (84). ICP0 cDNAs were inserted into a puromycin-resistant lentivirus backbone with the DCMV promoter and downstream TetO sites to create, for example, pLKO.DCMV.TetO.cICP0, which encodes the wt ICP0 protein. Derivatives of the wt ICP0-expressing lentivirus were constructed by exchanging the wt sequences with those including the FXE, D8, D12, D13, D15, and E52X deletion mutations, described previously (17, 61). All were based on the cDNA version of the ICP0 gene (21).

Lentivirus transduction. Lentivirus supernatants were prepared after cotransfection into HEK-293T cells of a pLKO series plasmid with pVSV-G (expressing the vesicular stomatitis virus envelope protein G) and pCMV.DR.8.91 (expressing all necessary lentivirus helper functions), as described previously (27). HepaRG cells were transduced with a lentivirus expressing the EGFPnlsTetR fusion protein to create HA-TetR cells by selection with G418 (initially 1 mg/ml and then reduced to 0.4 mg/ml during subsequent passage). HA-TetR cells were subsequently transduced with lentiviruses derived from the pLKO.DCMV.TetO.cICP0 series to create HA-cICP0, HA-cFXE, HA-cD8, HA-cD12, HA-cD13, HA-cD14, and HA-cE52X cells. These cells were selected with G418 and puromycin (initially 1 µg/ml, and then reduced to 0.5 µg/ml during subsequent passage). All cells were maintained with continuous antibiotic selection.

Induction of ICP0 expression. Cells were treated with medium containing tetracycline (catalog no. T7660; Sigma-Aldrich) at 0.1 µg/ml for various times as indicated in the text. For most experiments, the induction time was 16 to 24 h. Tetracycline was maintained in the medium throughout the duration of an experiment after the initial induction in order to maintain expression of ICP0.

Virus plaque, yield, and reactivation assays. For plaque assays, cells were seeded into 24-well dishes at 1 x 105 cells per well and then infected the following day with appropriate sequential threefold dilutions of in1863 or dl1403/CMVlacZ. After virus adsorption, the cells were overlaid with medium containing 1% human serum, and then the cells were stained for β-galactosidase-positive plaques 24 h later, as described previously (48). For virus yield assays, cells in 12-well dishes were infected with viruses at the MOIs stated in the text; 24 h later the cells were scraped into the medium, and the mixture was sonicated and clarified prior to titration on U2OS cells. For reactivation (derepression) assays, cells in 24-well dishes were infected with in1374 at an MOI of 5 PFU per cell and at nonpermissive temperature (38.5°C) and then incubated at the nonpermissive temperature for 24 h. Derepression of the lacZ marker gene in the in1374 genome was induced by treatment with tetracycline (0.1 µg/ml) for 24 h to induce ICP0 expression. Cells were stained for β-galactosidase activity the following day, using the blue plaque detection method noted above.

Infections and Western blot analysis. Cells were seeded into 24-well dishes at 1 x 105 cells per well. After the relevant experimental manipulations, the cell monolayers were washed twice with phosphate-buffered saline before being harvested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer. Proteins were resolved on 7.5% sodium dodecyl sulfate gels and then transferred to nitrocellulose membranes by Western blotting. The following antibodies were used: anti-ICP0 mouse monoclonal antibody (MAb) 11060 (21), anti-actin MAb AC-40 (Sigma-Aldrich), anti-PML MAb 5E10 (87), anti-Sp100 rabbit serum SpGH (85), anti-ICP4 MAb 58S (83), and anti-EGFP rabbit serum ab290 (Abcam).

Immunofluorescence and confocal microscopy. Cells on 13-mm glass coverslips were fixed and prepared for immunofluorescence as described previously (26). PML was detected with rabbit serum r8 or MAb 5E10, Sp100 was detected with rabbit serum SpGH, ICP4 was detected with MAb 58S, hDaxx was detected with rabbit serum r1866 (73), and ATRX was detected with rabbit serum H-300 (Santa Cruz Biotechnology, Inc). The secondary antibodies used were Cy3-conjugated goat anti-mouse immunoglobulin G and Cy5-conjugated goat anti-rabbit immunoglobulin G (GE Healthcare). The samples were examined using a Zeiss LSM 510 confocal microscope, with 488-nm, 543-nm and 633-nm laser lines, scanning each channel separately under image capture conditions that eliminated channel overlap. The images were exported as TIF files and then processed using Photoshop.


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RESULTS
 
Construction of an inducible cell line system for ICP0. Given the complexities of the various functions attributed to ICP0 (19, 42) and the difficulties inherent in utilizing ICP0-mutant HSV-1 strains (20), it is apparent that a cell line that expresses ICP0 in an inducible manner at close to physiological levels would be of great utility. Any system must be inducible since ICP0 expression at more than minimal levels is incompatible with long-term cell survival (22, 46, 49, 82). Such a system would avoid potential difficulties inherent in other expression methods, such as viral vectors (problems include expression of viral vector proteins and the effects of viral virion components on the infected cell) and transfection (limitations include variable frequency of positive cells and expression at very high, possibly nonphysiological levels in the transfected cells). We have developed a tetracycline-dependent induction (Tet-on) system built into lentivirus vectors to achieve this aim. The first component is a lentiviral vector that includes a hybrid gene driven by the HCMV promoter/enhancer comprising the tetracycline repressor (TetR) linked to an NLS and EGFP to produce an EGFPnlsTetR fusion protein. In addition, this vector encodes neomycin resistance (Fig. 1A). The second component is another lentivirus vector, this time with puromycin resistance, in which a fragment of the HCMV promoter/enhancer (DCMV) drives expression of the ICP0 cDNA (Fig. 1B). Tandem tetracycline operator (TetO) sequences have been inserted 10 bp downstream of the TATA box of the DCMV promoter in this vector (Fig. 1A). We also constructed variants of this lentivirus vector that include the mutant ICP0 cDNAs depicted in Fig. 1C.


Figure 1
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  		FIG. 1. Maps of the lentivirus plasmid vectors constructed to express the EGFPnlsTetR fusion protein (A) and wt ICP0 from a cDNA fragment under the control of a tetracycline inducible promoter (B). Panel C depicts the 775-residue ICP0 amino acid sequence, with the approximate locations of key domains marked. The shaded boxes below indicate the in-frame deletion mutations that are used in this study with the ranges of the residues that are deleted in mutants FXE, D8, D12, D13, D14, and E52X. USP7, USP7 binding domain; ND10, sequences required for efficient localization to ND10; LTR, long terminal repeat; RRE, Rev response element; hPGK, human phosphoglycerate kinase.

We chose to use HepaRG cells (36) for this series of experiments because this is a permanent cell line with minimal chromosomal aberrations that is a good host for HSV-1, and it is a line in which ICP0-null mutant strains exhibit a several hundredfold defect (29; see also further data below). Furthermore, these cells are readily transduced by lentiviruses. Initially, HepaRG cells were transduced with lentivirus prepared from plasmid pLKOneo.CMV.EGFPnlsTetR, and a neomycin-resistant mixed cell population (named HA-TetR) was isolated in which almost all cells expressed the EGFP fusion protein located in the nucleus. Next HA-TetR cells were transduced with lentivirus prepared from pLKO.DCMV.TetO.cICP0, and a mixed cell population (named HA-cICP0) expressing both neomycin and puromycin resistance was selected. HA-cICP0 cells were treated with tetracycline (0.1 µg/ml) for 24 h and then examined for ICP0 expression by immunofluorescence (Fig. 2). Before induction, out of 573 cells counted, 21 (3.7%) exhibited ICP0 expression at barely detectable levels, and 2 (0.35%) had stronger ICP0 staining. The remaining 96% of cells were negative. After induction, out of 280 cells counted, only 5 were negative for ICP0 expression, and 98% were positive. Time course experiments showed similar results at 16 h after induction, but prior to that cells began to express ICP0 in an asynchronous fashion. ICP0 expression could be detected as early as 4 h after induction, initially in a punctate pattern, with ICP0 colocalizing with PML in ND10 (data not shown). As time progressed, ICP0 was expressed in a greater proportion of cells, becoming progressively less punctate as PML staining was lost and ND10 proteins were disrupted (data not shown but see Fig. 5 below). These observations parallel the localization pattern of ICP0 expressed during HSV-1 infection, except that the accumulation of ICP0 in the cytoplasm that occurs at later times of infection (24, 50, 57) does not occur in uninfected HA-cICP0 cells. After induction, the cells could be maintained in the presence of tetracycline for up to 4 days before progressive cell loss led to degradation of the culture due to the long-term toxic effects of ICP0.


Figure 2
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  		FIG. 2. Analysis of cell line HA-cICP0 before (A and B) and after (C and D) induction of ICP0 expression by treatment with tetracycline (0.1 µg/ml) for 24 h. Samples on coverslips were stained for ICP0 using a Cy3-labeled secondary antibody (B and D). Panels A and C show EGFP autofluorescence.


Figure 5
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  		FIG. 5. Immunofluorescence analysis of cell lines induced to express wt and mutant ICP0 proteins at 24 h after induction. Each row shows a typical view of cells stained for PML, ICP0, EGFP autofluorescence, and the merged PML (green) and ICP0 (red) channels. The channels are shown in these colors to aid visualization of the merged images. The cell names are indicated on the right. In the cases of HA-cD12, HA-cD13, and HA-cE52X images, fields of view have been chosen to include cells expressing the ICP0 proteins at a variety of expression levels or to include cells not expressing the ICP0 protein for comparison of PML distributions in ICP0-positive and -negative cells.

HA-cICP0 cells allow complete complementation of ICP0-null mutant HSV-1. To test the functionality of ICP0 expressed after induction of HA-cICP0 cells, HSV-1 plaque assays were conducted in HA-cICP0, HA-TetR, and U2OS cells in parallel, before and after tetracycline treatment. Viruses in1863 and dl1403/CMVlacZ were used for the wt and ICP0-null mutant strains as these include an HCMV-driven lacZ gene that allows easy identification of plaques through staining for β-galactosidase. In the absence of tetracycline, the wt virus gave an average of 2.1- and 2.4-fold more plaques on HA-TetR and HA-cICP0 cells, respectively, than on U2OS cells stained at 1 day after infection. After tetracycline treatment, these ratios were 2.1- and 4.7-fold (data are summarized in Fig 3). These data indicate that in this series of experiments the HepaRG-based cells were slightly more permissive than U2OS cells for wt HSV-1 plaque formation and that preinduction of ICP0 increased the number of wt plaques by around twofold. For the ICP0-null mutant virus, the absolute number of plaques on HA-TetR cells was around 400-fold less than that on U2OS cells, which, taken with the twofold enhancement of wt HSV-1 in HA-TetR compared to U2OS cells, means that the number of plaques expected on HA-TetR cells if the mutant stock were wt was reduced by around 800-fold (Fig. 3 and Table 1). The absolute number of plaques of the mutant virus on HA-cICP0 cells before induction was around sixfold greater than on HA-TetR cells, presumably reflecting greater permissivity of the cells expressing low levels of ICP0 before induction. However, after ICP0 induction the titer of the mutant virus increased to levels expected, compared to that on U2OS cells, of a wt virus stock (Fig. 3 and Table 1). It is clear that HA-cICP0 cells allow complete complementation of ICP0-null mutant HSV-1.


Figure 3
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  		FIG. 3. Plaque formation efficiencies of wt HSV-1 (in1863; upper panel) and ICP0-null mutant HSV-1 (dl1403/CMVlacZ; lower panel) on U2OS, HA-TetR, and HA-cICP0 cells without or with prior induction of ICP0 expression using tetracycline (Tet) at 0.1 µg/ml for 24 h. Stocks of the two viruses were titrated on the cell lines in parallel, and then plaques were visualized the following day by staining for β-galactosidase activity. Mean titers of the wt and mutant virus stocks from two independent titration experiments are shown on log scales, with the error bars showing the range of values obtained.


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  		TABLE 1. Relative plaque-forming efficiencies of ICP0-null mutant HSV-1 in cell lines expressing wt and mutant forms of ICP0a

ICP0 expression in HA-cICP0 cells leads to extensive degradation of PML and loss of SUMO-modified Sp100 isoforms. Expression of ICP0 and its effects on major ND10 proteins PML and Sp100 were then tested in a time course experiment by Western blotting. HA-TetR and HA-cICP0 cells were treated with tetracycline, and parallel samples were harvested at various times thereafter. In HA-TetR cells the PML and Sp100 signals increased with time, but this was an effect of cell growth rather than the tetracycline treatment as the samples at 24 h with or without treatment were identical (Fig. 4). Expression of ICP0 could be detected in the induced HA-cICP0 cells by 8 h after induction by this method, increasing to a plateau at 16 h as the proportion of positive cells increased to the maximum (as discussed above). Other Western blot experiments indicated that the level of ICP0 at 24 h after induction was intermediate between that at 2 h and 4 h of wt HSV-1 infection at an MOI of 1 in HA-TetR cells (data not shown) (see Fig. 1 of the accompanying paper [28]). PML levels were drastically reduced in the HA-cICP0 samples at 16 and 24 h after induction, while Sp100 exhibited the relative loss of the lower-mobility isoforms coupled with an increase in the amount of the band corresponding to Sp100 isoform A unmodified by SUMO-1 (Fig. 4). These data are entirely consistent with earlier experiments using virus infection and other methods (3, 9, 10, 23, 38, 69, 71), but for the first time they demonstrate that degradation of PML and modification of Sp100, at the level of endogenous proteins as detected by Western blotting, can be achieved effectively by ICP0 without any contribution from other viral proteins.


Figure 4
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  		FIG. 4. Degradation of PML and loss of low-mobility isoforms of Sp100 induced by ICP0. Samples of HA-TetR and HA-cICP0 cells were treated with tetracycline at 0.1 µg/ml, and then whole-cell extracts were harvested at the indicated times after infection. ICP0, PML, Sp100, EGFPnlsTetR, and actin were analyzed by Western blotting.

Use of the inducible cell line system to analyze the functional domains of ICP0. The data presented above illustrate the utility of the inducible cell line system in determining functions of ICP0 in a straightforward manner. We next developed cell lines to express mutant forms of the protein in order to test the roles of specific regions of ICP0 in its core functions in lytic and reactivating infections. We chose to study a series of internal deletion mutants, namely, a RING finger deletion (FXE), a mutant lacking the NLS (D8), a mutant unable to bind to USP7 (D12), two mutants that remove sequences involved in complex functions including efficient ND10 localization (D13 and D14), and a mutant removing the entire C-terminal quarter of the protein (E52X) (Fig. 1C). A more detailed summary of the properties of these mutants is given below in the Discussion.

Although all these mutations have been studied previously using mutant viruses in a selection of assays (2, 9, 18, 23, 25, 45), additional functions have since been attributed to the C-terminal region (37, 39-41, 43), and not all have been studied in both lytic and reactivating infections. Furthermore, because the particle-to-PFU ratios of these ICP0 mutant viruses vary over wide ranges (18, 20), there can be difficulties in comparing their phenotypes because of variations in the proportions of cells infected at a given multiplicity and/or the need to use significantly different multiplicities of wt and mutant viruses in order to ensure that a high proportion of the cells in a population engage in a productive infection (20). The inducible cell line system enables the study of different ICP0 mutant proteins in experiments in which these complications no longer apply.

Immunofluorescence analysis of cells expressing wt and mutant ICP0 proteins. Lentiviruses including these mutant ICP0 genes were constructed and used to transduce HA-TetR cells to generate cells named HA-cFXE, HA-cD8, and so on. All were similar to HA-cICP0 cells in terms of very low numbers of cells expressing ICP0 prior to induction and very high proportions positive for expression after tetracycline treatment. We noted that ICP0 fluorescence intensities in induced HA-cD12 cells were weaker than in the other cells in this series, which is a reflection of reduced expression levels detected by Western blotting (see Fig. 6 below) and almost certainly a consequence of loss of USP7 binding and hence greatly decreased stability of this mutant protein (2, 9, 34).


Figure 6
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  		FIG. 6. Ability of wt and mutant forms of ICP0 to degrade PML and induce loss of the lower-mobility isoforms of Sp100. HA-TetR, HA-cICP0, and the analogous cell lines transduced with lentiviruses including mutant ICP0 genes were treated with tetracycline (Tet) at 0.1 µg/ml for 24 h or left untreated. Whole-cell extracts were prepared and analyzed by Western blotting for PML, Sp100, ICP0, and actin. (A) The results with HA-TetR, HA-cICP0, and cells expressing the FXE, D8, D14 and E52X mutants of ICP0 are presented. (B) The results with HA-TetR, HA-cICP0, and cells expressing the D12 and D13 mutant forms of ICP0 are presented. For this part, three separate blots were prepared, and the actin profile is shown for each. The lower intensity of the Sp100 bands in the HA-TetR sample in this particular gel is likely due to underloading (see the actin profile) as this was not observed in other similar analyses. The levels of the mutant ICP0 proteins as determined by densitometry were as follows: wt, 1.0; FXE, 1.0; D8, 0.84; D14, 2.1; E52X, 2.1; D12, 0.19; and D13, 2.3.

The effects of the various ICP0 proteins on ND10 integrity were studied by immunofluorescence at 16 h after induction (Fig. 5). Although PML was almost completely degraded in cells expressing wt ICP0 at this time point (Fig. 4), such cells retained small amounts of PML that were detectable by immunofluorescence, but this was invariably highly modified in distribution compared to control cells (Fig. 5, HA-TetR and HA-cICP0). In contrast to the colocalization of wt ICP0 and PML observed at early times after induction (noted above), any remaining PML rarely colocalized with any distinct ICP0 foci. As expected, neither the RING finger FXE deletion mutant nor the D8 mutant lacking the NLS affected ND10 structure, and in the case of the former there was considerable stable colocalization of PML and FXE mutant ICP0 (Fig. 5, HA-cFXE). As noted earlier, the D12 mutant gave reduced fluorescence intensities but nonetheless was expressed in sufficient amounts to cause disruption of ND10 (Fig. 5, HA-cD12), the extent of which was variable between cells and was usually less than that seen with the wt protein. The D13, D14, and E52X mutants gave similar results to each other, all presenting a largely nuclear diffuse staining pattern and having some disruptive effect on ND10, which was again variable between cells (Fig. 5).

Parallel samples of all cells were also analyzed by double staining for PML and other major ND10 proteins including Sp100, hDaxx, and ATRX (see Fig. 1 to 6 posted at ftp://gamma.vir.gla.ac.uk/pub/). In the wt ICP0-expressing cells, these other proteins were almost completely dispersed and rarely exhibited colocalization with any remaining PML foci. In cells expressing the FXE and D8 mutant proteins, all studied ND10 constituents colocalized in normal ND10 foci. In cells expressing cells D12, D13, D14, and E52X, there was variation in the extent of the changes in distribution of the proteins so that the extent of colocalization of the other ND10 proteins with any remaining PML foci varied not only between cells but also between different PML foci within a single cell. In general, any remaining Sp100 foci frequently colocalized with PML, whereas hDaxx and ATRX were more frequently dispersed. Therefore, we conclude that the deletion mutations in the C-terminal region of ICP0 give a partially defective phenotype in terms of ND10 disruption.

Only the RING finger and NLS domains of ICP0 are essential for degradation of PML. As noted above, there are complications in comparing the efficiency of PML degradation induced by mutant forms of ICP0 during virus infection since the mutants have very different particle/PFU ratios, and ensuring that all cells are equally infected is not a straightforward matter (20). The use of the inducible cell line system overcomes this problem. Samples of the series of mutant cell lines were harvested with or without induction of ICP0 expression and then analyzed for PML and Sp100 by Western blotting. Figure 6 shows that the levels of expression of the various ICP0 mutant proteins, with the exception of the D12 mutant, were between 0.84- and 2.3-fold of the wt protein (further details are included in the figure legend). The D12 mutant protein is unstable because of its failure to bind USP7 (9), and it therefore accumulates to significantly lower levels than wt ICP0. PML was degraded in the wt and all the mutant ICP0-expressing cell types save in HA-cFXE and HA-cD8 cells. PML degradation was slightly less efficient than in the case of wt ICP0 in cells expressing the D13, D14, and E52X proteins (despite their increased levels of accumulation) and was least efficient in HA-cD12 cells (consistent with the decreased levels of this mutant protein). With the exception of mutant D12, loss of the lower-mobility Sp100 isoforms and a relative increase in unmodified Sp100-A correlated with loss of PML. In the case of the HA-D12 cells, changes in the Sp100 profile were marginal despite significant degradation of PML. Although this experimental design does not allow a comparison of the rates at which the various ICP0 proteins induce degradation of PML and loss of selected Sp100 isoforms, we can conclude that nuclear localization and the RING finger domain of ICP0 are essential for these activities while all sequences in the C-terminal third of the protein are partially dispensable. These data broadly reflect the immunofluorescence analysis presented above.

Plaque assay complementation analysis of ICP0-null mutant HSV-1 in cells expressing mutant forms of the protein. Cells expressing the series of wt and mutant ICP0 proteins were used in plaque assays of wt (in1863) and ICP0-null mutant (dl1403/CMVlacZ) viruses with or without prior induction of ICP0 expression. With the exception of the slight increase in wt plaque numbers in HA-cICP0 cells after ICP0 induction noted earlier (Fig. 3), the wt virus gave closely similar titers in all cell types (data not shown). A summary of the titration results with the ICP0-null mutant virus in the various cell lines is presented in Table 1. In comparison with the wt protein, the RING finger mutant FXE and the NLS mutant D8 complemented to 0.1 and 0.4% of the levels of the wt, indicating that these domains are essential for ICP0 function. In contrast, deletion of the complete C-terminal region still allowed plaque formation at an efficiency of around 9% while the other smaller deletions in this region gave complementation levels varying between 10% and 40%. The data were compared with the efficiency of plaque formation of the cognate mutant viruses in HepaRG compared to U2OS cells, normalizing the data to the relative plaque formation efficiency of the wt virus in the two cell lines (Table 1). The results indicate a striking similarity between the two approaches. These data are broadly consistent with a previous independent study that found that an HSV-1 mutant expressing an ICP0 protein lacking the C-terminal 250 residues of ICP0 (a deletion roughly corresponding to the E52X mutant used here) was significantly more active than a complete deletion mutant in a variety of assays (8).

Analysis of the domains of ICP0 required for derepression of viral gene expression in a cultured cell model of HSV-1 quiescence. A critical function of ICP0 is its ability to reactivate gene expression from repressed viral genomes in cultured cell models of quiescent infection (45, 47, 64, 68, 80, 81, 86). Early work using an analogous but less tractable model indicated that the ICP0 RING finger was essential for reactivating quiescent HSV-2 but that the sequence deleted by the D14 mutation was not (45). These observations had not previously been extended to investigate the importance of other features of ICP0 in the cultured cell reactivation phenomenon. HSV-1 mutant virus in1374 carries a deletion of ICP0, a temperature-sensitive mutation in ICP4, a mutation in VP16 that renders it incapable of stimulating IE gene transcription, and a marker lacZ gene linked to the HCMV IE promoter/enhancer (78). It has been demonstrated that human fibroblasts can be infected with this or similar mutant viruses at a multiplicity such that most cells in the population contain viral genomes, but these rapidly become repressed, as exemplified by lack of expression of the marker gene (77, 78, 80). Expression of the marker gene can be reactivated (or derepressed) by subsequent exogenous expression of ICP0 using viral vectors (45, 77, 80). The ICP0-inducible cell line system allows a powerful extension of this technology to determine the features of ICP0 that are necessary for derepression of marker gene transcription.

Accordingly, we infected HA-TetR and the ICP0 derivative cells with in1374 at nonpermissive temperature to establish cultures of quiescently infected cells, and then ICP0 expression was induced with tetracycline. Figure 7 illustrates that induction of wt ICP0 expression resulted in the efficient appearance of cells expressing β-galactosidase from the marker lacZ gene, whereas cells expressing the RING finger and NLS deletion mutants did not. All of the mutant ICP0 proteins lacking sequences in the C-terminal third of the protein, including removal of this entire region, were positive in the derepression assay (Fig. 7). These data were quantified by capturing images of random fields of cells from each well, calculating the proportion of cells positive for β-galactosidase expression after induction of wt ICP0 expression, and then expressing the number of positive cells in the other cells lines as a percentage of that in HA-cICP0 cells (Fig. 8). The results clearly show that, on the basis of positive cell counts, deletions in the C-terminal third of ICP0 are less deleterious for ICP0 function in reactivation/derepression than for stimulation of plaque formation, and in fact these proteins have close to wt activity in the derepression assay. Clearly, the only domains of ICP0 that are essential for inducing derepression of gene expression are the RING finger and the NLS while the C-terminal third of ICP0 is almost completely dispensable. These data parallel a previous study of HSV-1 latency and reactivation in mice using viruses expressing C-terminal truncation mutations of ICP0 which demonstrated that such mutants reactivated at about 50% of the efficiency of the wt virus (7).


Figure 7
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  		FIG. 7. Derepression of gene expression from quiescent viral genomes by wt and mutant forms of ICP0. Cells were infected or not (mock) with virus in1374 at an MOI of 5 PFU per cell at the nonpermissive temperature, and then 24 h later the cells, as indicated in each panel, were treated with tetracycline (Tet; 0.1 µg/ml). The cells were stained for β-galactosidase activity the following day to detect reactivated transcription from the lacZ marker gene in the in1374 genome. Cells positive for reactivated transcription turn blue, while negative cells give a clear background. The experiment was performed with the indicated cells. cICP0, HA-cICP0; cFXE, HA-cFXE; cD8, HA-cD8; cD14, HA-cD14; cE52X, HA-cE52X; cD12, HA-cD12; cD13, HA-cD13.


Figure 8
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  		FIG. 8. Quantification of the extent of derepression depicted in Fig. 7. Four random fields of view of the wells in the tetracycline-treated cells in the experiments shown in Fig. 7 were photographed, and then positive and negative cells were counted (approximately 250 cells per view). The proportions of positive cells (blue in Fig. 7) in the induced wells were plotted with respect to the counts in the HA-cICP0 samples. The bars show mean results from the different fields of view counted, with standard deviations. The background level of positive cells (Fig. 7) in HA-TetR cells representing those in which expression of the marker gene was not initially repressed is 0.5%. This constitutes the limit of detection, meaning that the assay extends over a 200-fold range.

Analysis of the recruitment of PML to the sites of viral genomes during infection with mutant HSV-1 viruses. In recent years it has been established that a prominent event during the early stages of HSV-1 infection is the recruitment of PML and other ND10 proteins to sites in close association with viral genomes soon after they enter the nucleus (26, 31, 32). This process is detectable as early as 30 min after the addition of virus to cells (27) and is therefore one of the earliest events that can be detected during infection, prior even to detectable synthesis of ICP4. Since this cellular response to the viral genome is strongly counteracted by ICP0 (26, 31), it is possible that it underlies the rapid repression of HSV-1 gene expression that occurs in the absence of ICP0 activity. Therefore, we tested the efficiency of this process in cells infected with mutant viruses expressing the various forms of ICP0 utilized in the HA-cICP0 series of cell lines. As discussed in detail below, these experiments allowed correlations to be made between this and several other suggested functions of ICP0 and the quantified effects of ICP0 on stimulating lytic infection and derepression of quiescent genomes presented above.

The protocol followed was to infect HF cells at low MOIs with wt and mutant viruses and then to analyze the cells by immunofluorescence the following day, staining for ICP4 and PML. As described in previous publications, newly infected cells at the periphery of developing plaques are infected by large numbers of virus particles, and the locations of the newly infecting genomes can be detected by staining for ICP4. Because of the directional mode of infection and the manner in which viral capsids are transported to the nuclear envelope, the viral genomes form a characteristic arc-like pattern close to the nuclear rim. This asymmetric distribution allows unambiguous visualization of recruitment of PML and other cellular proteins to sites that are closely associated with the viral genomes. By screening for cells in the early stages of infection with a characteristic asymmetric distribution of ICP4 foci and then determining whether PML recruitment was strong, weak, or absent, we were able to quantify the differences between wt and mutant virus infections. Typical representative images are presented in Fig. 9.


Figure 9
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  		FIG. 9. Inhibition of recruitment of PML to the sites of viral genomes by wt and mutant ICP0 proteins during the early stages of HSV-1 infection. HF cells were infected at low MOIs with wt and a selection of mutant viruses expressing ICP0 proteins utilized elsewhere in this study. At 24 h after infection, the cells were fixed and stained for PML (red) and ICP4 (green). Representative images of cells close to the edge of developing plaques were selected to show the sites of parental viral genomes and early replication compartments detected by concentrations of ICP4 in distinct foci. Most images show more than one such cell to illustrate the range of phenotypes observed. Each vertical set of three panels shows the output from the red (uppermost) and green channels, with the merged image shown below. Some images include cells that are not detectably infected for comparison of the normal pattern of PML staining.

As in previous studies (26, 31), recruitment of PML in wt infections was detected only in a small minority of cells that were at the very earliest stages of infection (judged by the intensity of ICP4 staining) (5% weak and 95% absent; n = 20). In contrast, PML recruitment was readily evident in all ICP0-null mutant dl1403-infected cells with asymmetric ICP4 foci (100% strong; n = 20). The difference between the wt and dl1403 infections reflects the ability of ICP0 to counteract the recruitment process. Recruitment of PML was as pronounced as in the ICP0-null mutant infection in all RING finger mutant FXE-infected cells (100% strong; n = 20). The results with mutant viruses D12, D13, D14, and E52X were intermediate between the wt and the null and FXE mutants (Fig. 9). There was variability both at the cellular level (in that some cells exhibited recruitment while others did not) and the intracellular level (in terms of the prominence of the recruitment effect). In general, recruitment was most obvious in cells expressing very low levels of ICP4 (and presumably therefore low levels of ICP0). Of these four particular mutants, D12 appeared most active in impeding recruitment (45% weak and 55% absent; n = 20). Cells exhibiting at least some recruitment were more frequent than in D12 infections with viruses D13 (30% strong, 30% weak, and 40% absent; n = 20), D14 (20% strong, 50% weak, and 30% absent; n = 20), or E52X (40% strong, 35% weak, and 35% absent; n = 20). This could correlate with the ability of the D12 mutant protein to localize to ND10 more efficiently than the others.

Therefore, just as the forms of ICP0 with deletion mutations in the C-terminal region are able to induce degradation of PML to varied extents in the cognate inducible cell line series (Fig. 6), they are able to impede recruitment of PML (and most likely other ND10-associated proteins) to sites closely associated with viral genomes but not quite as effectively as the wt protein. As discussed in more detail below, this intermediate phenotype broadly correlates with the intermediate activities of these mutant ICP0 proteins in complementing the plaque-forming defect of ICP0-null mutant HSV-1.


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DISCUSSION
 
We describe here a simple and flexible system for constructing cell lines that express wt and mutant forms of ICP0. This efficient system allows study of the functions of ICP0 in a manner that avoids complications inherent in short-term plasmid or viral vector-based expression technologies or the use of HSV-1 mutants. The system enables expression of ICP0 at levels similar to those during the first 2 h of a normal infection in the great majority of cells in a population, thereby fully complementing the plaque-forming defect of ICP0-null mutant HSV-1. Plasmid transfection methods are limited by their inefficiency and the problem of nonphysiologically high levels of expression in those cells that do express the protein. Viral vector-based methods (e.g., adenoviruses and defective HSV-1 mutants) overcome some of these problems but cannot eliminate the effects of other proteins expressed from the vector viral genome and/or the effects of components of the virus particle itself on host pathways. The use of mutant HSV-1 viruses is powerful in a variety of situations, but in terms of studying the specific effects of ICP0 itself, they are notoriously difficult to control. This results from cell type differences in the requirement for ICP0, the multiplicity dependence of the requirement for ICP0, the nonlinearity of infection efficiency with respect to input multiplicity of ICP0 mutant viruses, the difficulties in ensuring that equal numbers of cells are infected in comparisons of wt and mutant infections, and the impossibility of achieving this aim with equal input of virus particles (20). Furthermore, because ICP0 stimulates infection, it can be difficult to separate the direct effects of ICP0 itself from those that arise from later events during infection. The influence of all the above factors also varies between different mutant viruses, depending on the severity of the ICP0 defect. In terms of studying the direct effects of ICP0 itself and quantifying the relative defects of different mutant forms of the protein, the system described here offers many distinct advantages.

There are, however, some limitations of the system that should be recognized. The first is that the level of expression cannot be regulated reliably by varying the amount of tetracycline or time of induction. This is because decreasing these variables results in a reduction in the proportion of positive cells as well as the amount of ICP0 expressed, while increasing them does not augment ICP0 expression further. Therefore, dose-response experiments are not practicable. The second limitation is that most of our experiments have been conducted only after a period sufficient to induce ICP0 expression in the whole population, and therefore the timing is different from that of a natural infection.

This study has utilized the inducible cell system to quantify the ability of mutant forms of ICP0 to complement initiation of lytic infection, as measured by efficiency of plaque formation, and to derepress transcription of the viral genome in quiescently infected cells. The approach allows accurate and reliable quantification without the difficulties stemming from the use of virus mutants outlined above. In this way, we were able to compare the functional effectiveness of ICP0 mutants lacking the RING finger, the NLS, and various sequences within the C-terminal third of the protein in a more comprehensive and rigorous way then previously achieved. The data clearly show that the RING finger and the NLS are essential for ICP0 function in both lytic infection and derepression assays. Furthermore, these mutants were unable to degrade PML, disrupt ND10, modify Sp100, and (in the case of the RING finger mutant; the NLS mutant was not tested) impede recruitment of PML to the sites of viral genomes. These results are consistent with and extend previous findings (18, 23, 24, 26, 31).

The analysis of the roles of sequences in the C-terminal third of ICP0 presents a more complicated picture. A number of functions have been attributed to the C-terminal third of ICP0, including binding to USP7 (also known as HAUSP) (25), self-multimerization (11, 61), efficient localization to ND10 (24), providing a second ubiquitin ligase domain named HUL-1 (41), and disruption of the HDAC1/CoREST complex through binding to CoREST (37). The results of previous studies on these functions are summarized in Fig. 10, along with a summary of the current results. These issues will be considered in turn.


Figure 10
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  		FIG. 10. A summary of the properties and functions of ICP0 that are relevant to the analysis in this paper. (A) The columns refer to PML degradation (degradn) after ICP0 induction (Fig. 6), loss of low mobility isoforms of Sp100 (de-modifn) (Fig. 6), disruption of ND10 as detected by PML immunofluorescence (IF disruptn) (Fig. 5), disruption of ND10 as detected by Sp100, hDaxx and ATRX immunofluorescence (see Fig. 1 to 6 posted at ftp://gamma.vir.gla.ac.uk/pub/), self-interaction/ability to form multimers (taken or predicted from references 11 and 61), stability of ICP0 (2, 9, 34), predicted ability to confer HUL-1 activity (domain defined as including residues 543 to 680; 41), and predicted ability to bind to Co-REST (binding domain defined as residues 668 to 718) (39, 40). The scoring system is as follows: ++, wt or close to wt activity or presence of relevant domain; +, positive but reduced activity; +/–, greatly reduced activity; –, no activity. Further qualification of the positive (+) scores is given in the text. (B) The columns refer to the presence of the RING finger domain, presence of the NLS, ability to localize to ND10 (locn) efficiently during HSV-1 infection and/or transfection (24), ability to disrupt ND10 (a summary of the analysis of panel A), ability to inhibit (inhibn) recruitment of PML to ICP4-associated sites (Fig. 9), ability to bind to USP7 (25, 61, 62), presence of HUL-1 and CoREST domains (as in panel A), complementation (complementn) of plaque formation of dl1403 in the various cell lines (Table 1), and efficiency of derepression of quiescent genomes (Fig. 7 and 8). The scoring system is the same as in panel A. ND, not done.

ICP0 mutant D12 is unable to bind to USP7 and in consequence becomes very unstable due to failure to protect against autoubiquitination (9). For reasons that are unclear but which may be connected with a loss of the self-interaction region, deletions that remove sequences further C-terminal to the USP7 binding site increase ICP0 stability, whether or not USP7 binding ability is retained (34). This property probably explains why the D13, D14, and E52X mutant proteins accumulate to much higher levels than the D12 mutant (Fig. 6). However, the quantity of D12 ICP0 that was expressed was sufficient to cause considerable disruption of ND10 (Fig. 5) although the effects at the level of PML degradation and, in particular, Sp100 modification were less dramatic (Fig. 6). The D12 mutant was also able to impede recruitment of PML to the sites of viral genomes with close to wt efficiency (Fig. 9) in that cells with prominent or even marginal recruitment were rare and certainly less common than in infections with the D13, D14, and E52X viruses. Complementation of ICP0-null mutant HSV-1 plaque formation by D12 ICP0 was the most efficient of all the mutants tested, consistent with the conclusion of a previous study that only small amounts of ICP0 are required to stimulate plaque formation (47). We note that the D12 mutant protein is able to localize to ND10 efficiently, whereas the D13, D14, and E52X proteins do not prominently colocalize with ND10, despite their obvious effects on ND10 structure. The D12, D13, D14, and E52X C-terminal deletion mutants were similarly active in the derepression assay (Fig. 7 and 8). This result is perhaps surprising in view of the inefficient reactivation of latent integrated adeno-associated virus genomes induced by the D12 mutant compared to the wt protein (34, 35). However, in an isolate of HA-cD12 cells that expressed lower levels of the protein than those studied here, derepression efficiency was at only 22% of the wt levels, suggesting that the amount of the protein expressed is an important factor in this assay.

Mutants D13, D14, and E52X all affect regions that are required for several reported functions of ICP0, namely, efficient localization to ND10, self-multimerization, HUL-1 activity, and binding to CoREST (24, 25, 37, 39-41, 43, 61, 71). Based on published mapping studies, all three of these mutants would be predicted to be negative for the HUL-1 and CoREST binding activities. All these mutant proteins retain significant activity in both lytic infection and derepression assays (Table 1 and Fig. 8 and 9). In the case of the former assay, they have activities that are around 2 orders of magnitude greater than the RING finger deletion mutant in an assay that covers a range of 3 orders of magnitude (Table 1). In the latter, they have activities again about 2 orders of magnitude greater than the RING finger deletion mutant in an assay that has a 200-fold range (Fig. 9 and 10). As noted above, removal of these C-terminal sequences of ICP0 did not inactivate the protein in a variety of assays in independent studies, including virus reactivation from latency in a mouse model (7, 8). Manifestly, the HUL-1 and CoREST binding activities are neither sufficient nor absolutely necessary for significant ICP0 activity during lytic infection and especially in the derepression assay.

Because of its implications in chromatin metabolism and the regulation of viral gene expression, the potential interaction between ICP0 and CoREST has attracted much recent attention. Accordingly, the significance of these observations merits careful analysis. The first study of this issue reported that CoREST could be detected in ICP0 immunoprecipitates at 7 and 14 h after infection (but not at 2 h) and that the HDAC1/CoREST complex was disrupted by wt HSV-1 at 14 h after infection but not at 7 h (37). Disruption of the HDAC1/ CoREST complex was less efficient in cells infected with an ICP0-null mutant virus (37). Later studies mapped the CoREST binding region to a segment overlapping the D13 and D14 deletions described in the current study although the authors noted that only a small proportion of CoREST could be coprecipitated with ICP0 from infected cells. Although the hypothesis that ICP0 might function through its effects on a known repression complex is attractive, the data presented above demonstrate that mutant ICP0 proteins that lack the reported CoREST interaction region function 2 orders of magnitude more efficiently than the RING finger deletion mutant in both lytic infection and derepression assays. While it remains possible that an interaction with CoREST plays a role in ICP0 function, it is clearly neither essential nor the major factor.

Notwithstanding the above, recent studies have demonstrated a correlation between ICP0 expression and an increased proportion of acetylated histones on viral chromatin during lytic and reactivating infections (12, 13). In a similar vein, earlier work had reported an association between class I and II HDACs in cells expressing exogenous ICP0 and the HDACs at high levels and an interaction between class II HDACs and ICP0 in glutathione S-transferase pull-down assays (56). However, ICP0 expression did not increase global acetylation levels of histone H4 during HSV-1 infection (56). While there is considerable and accumulating evidence that chromatin assembly and status play important roles in HSV-1 biology, particularly during establishment, maintenance, and reactivation from latent infections (1, 13, 14, 51, 52, 88, 89), it is important to consider whether any changes in histone acetylation patterns during lytic infection are due directly to ICP0 activity or are an indirect consequence resulting from the increased transcriptional activity that occurs in the presence of ICP0. If the principle role of ICP0 were to interfere with HDAC activities, it would be expected that alternative methods of inhibiting HDAC enzymes would circumvent the need for ICP0. While the HDAC inhibitor trichostatin A can increase lytic infection, derepression, and reactivation from latency to a certain extent in some experimental systems (1, 14, 63, 74, 88), the drug had no effect on the replication efficiency of ICP0-null mutant HSV-1 in either human fibroblasts or HepaRG cells (29) and does not augment derepression of quiescent HSV-1 genomes in fibroblasts (70, 78, 88).

Another hypothesis to explain the repression of HSV-1 gene expression that occurs in the absence of ICP0 is that the rapid recruitment of PML and other ND10 proteins to the sites of parental viral genomes represents the core of a cellular antiviral response. This by no means excludes the possibility that chromatin assembly events are involved in viral genome repression. The recruitment phenomenon is one of the earliest observable events in an infected cell, and it can be detected (in the absence of ICP0) within 30 min of adding the virus to the cell monolayer, certainly before detectable viral gene expression (27). One role of ICP0 is rapidly and efficiently to eliminate this recruitment (26). We have reported that depletion of PML and Sp100 individually increases replication efficiency of ICP0-null mutant HSV-1 (29, 30, 33) and that simultaneous depletion of both of these cellular proteins increases mutant virus plaque formation yet further (29). However, it was not possible by these procedures to overcome completely the need for ICP0, and indeed the cell retains a substantial proportion of its intrinsic ability to repress HSV-1 gene expression even in the absence of PML (29, 30). Nonetheless, we note that neither PML nor Sp100 is required for the recruitment of other ND10-related proteins to the sites of viral genomes (29, 30), so although PML and Sp100 contribute to the intrinsic cellular antiviral response, other major factors remain to be defined.

As a working hypothesis, we propose that the mechanism by which PML and other proteins are recruited to the sites of viral genomes is an important, perhaps essential, part of the cellular intrinsic defense to DNA virus infection. ICP0 overcomes this response in a RING finger-dependent mechanism that correlates with the degradation of PML and the disruption of ND10. We note that the mechanism by which PML is degraded by ICP0 may also apply to other cellular proteins, and we suggest that this process (and not necessarily the degradation of PML itself) is essential for eliminating the recruitment phenomenon. This hypothesis is consistent with the key properties of ICP0 that are summarized in Fig. 10 in that ICP0 mutants that do not inhibit recruitment are inactive, whereas those that are partially active in lytic infection and derepression assays exhibit partial activities in the recruitment assay. This hypothesis predicts that a defect in the cellular recruitment response will, at the least, decrease the need for ICP0 for efficient HSV-1 infection. This model is currently under investigation.


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ACKNOWLEDGMENTS
 
This work was supported by the Medical Research Council and in part by EC FP6 SME-STREP project TargetHerpes (LSHG-CT-2006-037517). M.-L.P. was a project student from ENS Lyon during her work in the MRC Virology Unit in the Everett laboratory.

Chris Preston supplied viruses in1863, dl1403/CMVlacZ, and in1374. We thank Roel van Driel (Amsterdam, The Netherlands) and Hans Will (Hamburg, Germany) for generous provision of anti-PML and anti-Sp100 antibodies, Huseyin Sirma (Hamburg, Germany) for HepaRG cells, and Rick Randall (St. Andrews, United Kingdom) for advice on interferon-related experiments. We thank members of the R.D.E. group for stimulating discussions throughout the course of this project and Chris Preston and Chris Boutell for constructive criticism of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, Scotland, United Kingdom. Phone: 44 141 3398855. Fax: 44 141 3372236. E-mail: r.everett{at}mrcvu.gla.ac.uk Back

{triangledown} Published ahead of print on 4 March 2009. Back


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Journal of Virology, May 2009, p. 4963-4977, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02593-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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  • Everett, R. D., Orr, A. (2009). 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. J. Virol. 83: 4978-4983 [Abstract] [Full Text]  

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