STAT-1- and IRF-3-Dependent Pathways Are Not Essential for Repression of ICP0-Null Mutant Herpes Simplex Virus Type 1 in Human Fibroblasts

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1 JOURNAL OF VIROLOGY, Sept. 2008, p Vol. 82, No X/08/$ doi: /jvi Copyright 2008, American Society for Microbiology. All Rights Reserved. STAT-1- and IRF-3-Dependent Pathways Are Not Essential for Repression of ICP0-Null Mutant Herpes Simplex Virus Type 1 in Human Fibroblasts Roger D. Everett, 1 * Dan F. Young, 2 Rick E. Randall, 2 and Anne Orr 1 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, Scotland, United Kingdom, 1 and School of Biology, University of St. Andrews, The North Haugh, St. Andrews KY16 9ST, Scotland, United Kingdom 2 Received 19 March 2008/Accepted 19 June 2008 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. Downloaded from One of the major mechanisms by which cells resist virus infection is through a complex set of related pathways involving secreted cytokines known as interferons (IFNs) (reviewed in reference 39). The binding of IFNs to receptors on the cell surface initiates signal transduction processes, which lead to the transcriptional activation of a large number of IFN-stimulated genes (ISGs), many of which have established antiviral functions (reviewed in reference 39). This process involves the phosphorylation of STAT-1, followed by its translocation to the nucleus and its participation in a transcriptional activation complex known as ISGF3 that assembles on the IFN-stimulated response elements (ISREs) within the promoters of ISGs. Accordingly, cells that lack STAT-1 are unable to stimulate ISG expression in the presence of IFN (reviewed in reference 39). Cells respond to virus infections by the recognition of pathogen-associated molecular patterns (including, for example, double-stranded RNA), which activate signal transduction mechanisms that lead to the activation of IFN- gene transcription, principally through the assembly of a transcriptional activation complex containing IFN regulatory factor 3 (IRF-3) on the IFN- gene promoter (reviewed in reference * Corresponding author. Mailing address: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, Scotland, United Kingdom. Phone: Fax: r.everett@mrcvu.gla.ac.uk. Published ahead of print on 25 June ). Viruses have evolved a wide variety of mechanisms to evade the IFN response, among which are the degradation of STAT-1 to eliminate IFN signaling or of IRF-3 to inhibit IFN production (reviewed in reference 39). Herpes simplex virus type 1 (HSV-1) infection is sensitive to both type I (IFN- / ) and type II (IFN- ) IFNs, and the two types can act synergistically to inhibit virus gene expression at a variety of different stages, including the repression of immediate-early (IE) gene expression (17, 27, 31, 33, 34, 41). IFNs also play a key role in regulating the pathogenesis of HSV-1 in mouse models (18, 22). Of particular interest for the study reported here is the potential link between the IFN-mediated repression of HSV-1 gene expression and the functions of ICP0, a key viral IE regulatory protein that plays a critical role in the regulation of HSV-1 infection (for reviews, see references 10 and 16). Viruses that fail to express ICP0 have a markedly reduced probability of establishing a lytic infection in human fibroblasts (HFs) infected at a low multiplicity because their genomes are highly susceptible to repression. This phenotype is cell type dependent, being most prominent in primary HFs, of medium severity in commonly used transformed cell lines such as Vero and BHK cells, and absent in osteosarcoma U2OS cells (11, 43, 46). The work described in this report was stimulated by previous findings that (i) ICP0-null mutant HSV-1 is highly sensitive to IFN treatment of Vero cells (19, 29, 30); (ii) the IFN sensitivity of ICP0-null mutant HSV-1 is dependent on the presence of on April 28, 2018 by guest 8871

2 8872 EVERETT ET AL. J. VIROL. the cellular ND10 promyelocytic leukemia (PML) protein in mouse fibroblasts (4); (iii) that high-multiplicity infection of HFs with highly defective HSV-1 mutants induces the IFNindependent induction of ISGs (9, 28, 32, 37); (iv) ICP0 interferes with defective HSV-1 induction of ISGs, and IRF-3 stimulated ISG transcription (9, 23, 25, 26); and (v) ICP0-null mutant HSV-1 becomes as pathogenic as the wild-type (wt) virus in STAT-1 knockout mice (18). These observations suggest that the low probability of plaque formation of ICP0-null mutant HSV-1 in HFs may be due to a failure to inactivate the repression of HSV-1 gene expression through a pathway related to an IFN response or ISG induction. The work described in this paper was designed to explore this hypothesis. It should be emphasized that this is a complex issue with the potential involvement of several distinct processes including (i) IFN-induced gene expression through IFN receptors, STAT- 1-dependent signaling, and the assembly of ISGF3 complexes on ISREs in ISG promoters; (ii) multiple pathways that lead to IRF-3 activation and stimulation of IFN- gene transcription; and (iii) enhancement of ISG expression through the IFNindependent induction of complexes on the ISREs of ISG promoters that contain IRF-3 but are distinct from ISGF3 (9, 28, 37). As noted above, ICP0 has been found to interfere with the latter two processes in certain experimental situations (9, 23, 30). Furthermore, distinction must be made between overcoming an existing IFN response (as in IFN pretreatment experiments) and the inhibition of induction of ISG expression during infection of untreated cells. The aim of these experiments was not to determine how IFN restricts HSV-1 infection, nor to challenge any of the abovedescribed findings, but only to establish if any of the abovedescribed phenomena play a key role in the low probability of plaque formation by ICP0-null mutant HSV-1 in HFs. We report that (i) neither STAT-1 nor IRF-3 is essential for the ICP0-null mutant plaque-forming defect in HFs, (ii) the extent of inhibition of plaque formation by ICP0-null mutant HSV-1 by IFN- in HFs is only about fivefold greater than that of the wt virus, (iii) the IFN sensitivity of both wt and ICP0-null mutant HSV-1 in HFs is hugely dependent on the multiplicity of infection (MOI), (iv) the IFN sensitivity of wt and ICP0-null mutant HSV-1 in HFs is not dependent on normal levels of PML, and (v) the extensive depletion of PML increases both wt and ICP0-null mutant HSV-1 plaque formation efficiency in IFN-treated HFs by around threefold and fivefold, respectively. The latter factor is similar to the increase in levels of mutant virus plaque formation in untreated PML-depleted HFs, compared to control cells (14, 15). We conclude that STAT-1 and IRF-3 are not essential for the repression of ICP0-null mutant HSV-1 gene expression in HFs, and we suggest that IFN and PML are involved in separable mechanisms of repression of HSV-1 gene expression. MATERIALS AND METHODS Viruses and cells. HSV-1 strain 17 was the wt strain used, from which the ICP0 null mutant dl1403 was derived (44). Viruses in1863 and dl1403/cmvlacz are derivatives of the above-described virus that contain the lacz gene under the control of the human cytomegalovirus promoter/enhancer inserted into the tk gene (kindly provided by Chris Preston). Virus in1374 is deleted for ICP0, is temperature sensitive for ICP4, and has a mutation that inactivates the transcriptional activation function of VP16 (38). All viruses were grown in BHK cells (at a permissive temperature and in the presence of 3 mm hexamethylene bisacetamide in the case of in1374) and titrated in U2OS cells in which ICP0 is not required for the efficient replication of HSV-1 (46). All viruses were used at MOIs on the basis of their PFU titers in U2OS cells regardless of the cell type used (11). U2OS cells were grown in Dulbecco s modified Eagle s medium supplemented with 10% fetal calf serum. Baby hamster kidney (BHK) cells were grown in Glasgow modified Eagle s medium supplemented with 10% newborn calf serum and 10% tryptose phosphate broth. Primary HFs isolated from human foreskin tissue (HF cells) (Department of Urology, University of Erlangen) were grown in Dulbecco s modified Eagles medium supplemented with 10% fetal calf serum. All cell growth media were supplemented with 100 units/ml penicillin and 100 g/ml streptomycin. Lentiviral expression of short hairpin RNAs (shrnas) to deplete STAT1, IRF3, and PML from HFs. Genes encoding the V protein of parainfluenza virus type 5 (PIV5) (previously known as SV5) or NPro of bovine viral diarrhea virus (BVDV) were cloned from plasmids described previously (8, 20) into a bicistronic lentivirus vector derived from phr-sin-csgw (7). The resulting plasmids, pdl.piv5/v.w3.puro and pdl.bvdv/npro.puro, were used to generate recombinant lentiviruses that were subsequently used to engineer HF cell lines to constitutively expressed the V protein of PIV5 or NPro of BVDV. Briefly, the lentiviruses were generated by transfecting 293FT cells (Invitrogen) with the lentivirus vector plasmids together with plasmids pcmvr8.91 (that expresses the gag/pol, tat, and rev genes of human immunodeficiency virus) and pmd_g (that expresses the envelope gene of vesicular stomatitis virus). Two days after transfection, the culture media containing the lentiviruses were harvested, filtered through a m membrane filter, and stored at 70 C. These stocks were used to transduce HFs with subsequent puromycin selection, as described below. The resultant cells were named HF-PIV5V and HF-BVDVN cells, respectively. To deplete PML, HF cells were transduced with lentivirus vectors, prepared as described previously (13), expressing either control anti-luciferase or anti-pml shrnas (13) to produce HF-shLuci and HF-shPML1 cells, respectively. Transduced cells were selected with puromycin (initially 1 g/ml and then reduced to 0.5 g/ml during subsequent passage) and maintained in medium containing puromycin. Plaque assays in transduced cell lines. Cells were seeded into 24-well dishes at 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 the cells were then stained for -galactosidase-positive plaques 24 h later, as described previously (21). Relative probabilities of plaque formation were calculated by comparing the numbers of plaques on the different cell lines at each separate dilution of virus. This approach overcomes the problem of the highly nonlinear nature (with respect to virus dilution) of plaque formation by ICP0- null mutant viruses in HFs (11, 43, 44). For IFN treatment experiments, medium containing human IFN- (catalog number ; Calbiochem) at the stated concentrations was added to the cells 1 day after seeding, and plaque assays were then initiated the following day. After virus adsorption, cells were overlaid with medium containing IFN- at the same concentration as that during pretreatment. IFN assays. The amount of IFN secreted by cells was estimated by using a biological assay in Vero cells (1). Culture supernatants from infected cells were harvested, centrifuged at 20,000 rpm for 2 h to pellet progeny HSV-1, UV treated to inactivate residual virus, and then added to cells for 24 h before infection with encephalomyocarditis virus (0.05 PFU/cell). The development of a cytopathic effect was monitored 36 h postinfection. Alternatively, a commercial enzyme-linked immunosorbent assay for human IFN- (PBL Biomedical Laboratories) was used. This method was compromised, as culture medium was found to interfere with detection in the IFN standard samples. Western blot analysis. Cells were seeded into 24-well dishes at cells per well 1 day before initiating an experiment. Where necessary, IFN- treatment was carried out as described above. For virus infection experiments, the cells were infected with wt HSV-1 strain 17 or dl1403 at multiplicities indicated in the figure legends, and samples were then prepared at various time points thereafter. Whole-cell extracts were prepared by washing the cells twice with phosphate-buffered saline before harvesting in sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer. Proteins were resolved on 7.5% (or 12% for ISG15) sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose membranes by Western blotting. ICP0, ICP4, and actin were detected using anti-icp0 mouse monoclonal antibody (MAb) 11060, anti- ICP4 MAb 58S, and anti-actin MAb AC-40 (Sigma-Aldrich) as previously described (11). STAT-1, IRF-3, and ISG15 were detected with rabbit polyclonal antibodies pab (catalog number ; BD Biosciences), FL-425 (sc-9082;

3 VOL. 82, 2008 STAT-1- AND IRF-3-INDEPENDENT REPRESSION OF HSV Santa Cruz Biotechnology, Inc.), and H-150 (sc-50366; Santa Cruz Biotechnology, Inc.), respectively. Virus growth curves and yield experiments. Cells were seeded into 12-well dishes at a density of cells per well. The cells were treated as appropriate with IFN- for 24 h before infection or infected the following day with wt or ICP0-null HSV-1 at multiplicities indicated in the figure legends. At the indicated time points, the cells were scraped into the medium, and the mixtures were sonicated in a sonibath before titration on U2OS cells. Note that IFN treatment has little effect on either wt or ICP0-null mutant plaque formation in U2OS cells (30), so the results would not be influenced by any carryover of IFN. RESULTS Isolation of HFs deficient in STAT-1. Given the previous work on the IFN sensitivity of HSV-1 and the hypersensitivity of ICP0-null mutant HSV-1 to IFN in Vero cells, it is a reasonable hypothesis that the repression of ICP0-null mutant HSV-1 gene expression in HFs could be mediated through IFN-related effects. This hypothesis was supported by the finding that ICP0-null mutant HSV-1 is as pathogenic as the wt virus in STAT-1-deficient mice, whereas the mutant is relatively nonpathogenic in normal mice (18). To test whether this hypothesis applied to the replication of the virus in HFs, we made use of a lentiviral vector that expresses the V protein of PIV5 (previously known as SV5), which targets STAT-1 for efficient proteasome-mediated degradation (8; reviewed in reference 39). In preliminary experiments, we found that compared to control naïve cells, HFs transduced with the PIV5 V protein-expressing lentivirus express no detectable STAT-1 (Fig. 1A), give similar numbers of plaques after infection with wt HSV-1 (Fig. 1B), and are unable to mount an anti-hsv-1 response after pretreatment with IFN- at concentrations as high as 500 units/ml (Fig. 1C). These results show that HF- PIV5V cells are completely defective in the STAT-1 signaling pathway. A notable feature of these data is that wt HSV-1 is markedly more sensitive to IFN pretreatment in HFs than in Vero cells (compare Fig. 1C with data presented in references 19 and 29). ICP0-null mutant HSV-1 does not have an improved plaqueforming efficiency in HFs deficient in STAT-1. We next compared the relative plaque-forming efficiencies of wt and ICP0- null mutant HSV-1 on naïve HFs and HF-PIV5V cells depleted of STAT-1. In neither case did the depletion of STAT-1 result in a significant alteration of plaque-forming efficiency (Fig. 2A). Therefore, the repression of ICP0-null mutant HSV-1 gene expression that occurs in HFs, and which is the root cause of its severe defect in plaque formation, is not relieved by the inactivation of STAT-1-mediated IFN signaling. This phenomenon was investigated further in growth curve experiments. The two cell types were infected with wt or ICP0- null mutant HSV-1 at an MOI of 0.2 PFU per cell, and wells were then harvested for the titration of virus yields at various times thereafter. As expected, the wt virus replicated more rapidly than the mutant in normal HFs, with a yield about 2 orders of magnitude greater at 16 h postinfection (Fig. 2B and C). This differential decreased with time as the wt yields approached a plateau, while the mutant virus continued to spread through the cell monolayer. Levels of replication of the wt virus were similar in normal and STAT-1-depleted HFs (Fig. 2C). There was little difference in the ICP0-null mutant virus yields at the 16- and 24-h time points in the presence or absence of STAT-1 (Fig. 2B). At the 48-h time point, however, FIG. 1. Characterization of HFs expressing PIV5 V protein. (A) Western blot comparison of STAT-1 expression in naïve HFs compared to that in HF-PIV5V cells. The positions of the molecular weight markers are shown on the right. (B) wt HSV-1 expressing -galactosidase from a lacz reporter gene (in1863) was titrated in HF and HF-PIV5V cells in parallel. The dilution of virus used is shown. The plaques are stained blue, while in the absence of counterstain, the wells without plaques are clear. (C) HF and HF-PIV5V cells were pretreated with the indicated concentration of IFN- for 24 h prior to in1863 plaque assay using the same 10 4 dilution as in B. IFN pretreatment gives substantial reductions in plaque formation in HFs, but this does not occur in HF-PIV5V cells. the mutant virus yield in the STAT-1-depleted cells was enhanced compared to that in the controls. The extent of this enhancement varied between experiments, being 5-, 11-, 26-, and 52-fold in the four experiments summarized in Fig. 2B. Taken with the data shown in Fig. 2A, this suggests that while the mutant virus plaque formation efficiency is not enhanced by the loss of STAT-1 signaling, the spread of the mutant virus through the cell sheet might be impeded if the IFN signaling pathway is intact and the cells are able to produce and respond to IFN. To test this hypothesis, we attempted to measure IFN production in wt and ICP0-null mutant infections of HFs and HF-PIV5V cells at a variety of multiplicities (10-fold steps from 1 to 0.001) at the 16-h time point. We used both a commercial enzyme-linked immunosorbent assay and a biological assay (1), but we detected no increase in IFN above background levels in the culture medium in any of the infections (data not shown). This may have been due to limitations in the assays used, especially as IFN production has been detected after wt HSV-1 infection of mouse fibroblasts (35, 40). None-

4 8874 EVERETT ET AL. J. VIROL. FIG. 2. (A) Relative plaque-forming efficiencies of wt and ICP0- null mutant HSV-1 in STAT-1-depleted and naïve HFs were determined using viruses in1863 and dl1403/cmvlacz using a series of threefold virus dilutions. Plaque numbers in HF-PIV5V cells were expressed as a proportion of those in HFs obtained with the same virus dilution. The wt and ICP0-null mutant data are the averages of five and nine determinations, respectively, pooling the data from the dilutions series from independent experiments. Error bars are standard deviations (SD). (B) ICP0-null mutant virus yield time course in HF-PIV5V cells and naïve HFs. Cells were infected at an initial MOI of 0.2 PFU per cell, and replicate samples were then harvested at the indicated time points. The data are the averages of three independent experiments, and error bars are SD. (C) Same as described above (B) except that wt HSV-1 was used. (D) Induction of ISG15 during wt (strain 17 ) and ICP0-null (dl1403) infection. HFs were infected at an MOI of 1 PFU/cell, and samples were harvested at the indicated times of theless, there was a clear induction of ISG15 expression following infection of HFs with ICP0-null mutant HSV-1 but not in the parallel wt infection (Fig. 2D). ISG15 is a ubiquitin-like protein that can be conjugated to other proteins and is expressed from a well-characterized IFN-induced gene (24). The induction of ISG15 expression did not occur in HF-PIV5V cells after treatment with IFN or infection with multiply defective HSV-1 (Fig. 3C). Therefore, while STAT1-dependent signaling does not underlie the initial plaque-forming defect of ICP0-null mutant HSV-1 (i.e., the frequency at which an initially infected cell commits to lytic infection), it is possible that the induction of ISGs in normal HFs could limit ICP0-null mutant spread through an infected cell monolayer, thus accounting for the difference in the yields of the mutant virus in HFs and HF-PIV5V cells at later time points. Note that while it appears that the expression of ICP0 inhibits ISG induction (Fig. 2D), this is not necessarily the case. Under these infection conditions (MOI of 1 in HFs), the great majority of cells will have been productively infected with the wt virus, yet in the case of the mutant, only a minority of cells would have initially committed to the lytic cycle (11). It is possible that ISG15 synthesis is taking place in the apparently uninfected cells that have not committed to the lytic cycle rather than in the cells in which the ICP0-null mutant is replicating. ICP0-null mutant HSV-1 does not have an improved plaqueforming efficiency in HFs deficient in IRF-3. The above-described results indicate that the plaque-forming defect of ICP0-null mutant HSV-1 in HFs cannot be attributed to the induction of IFN synthesis followed by STAT-1-dependent signaling resulting in the expression of ISGs. However, it is not necessary to invoke the IFN-mediated induction of ISGs because it has been established that HSV-1 mutants that are highly defective in IE gene expression induce the expression of ISGs (9, 28, 32) through an IFN-independent pathway that is not dependent on STAT-1 (32). This process can be inhibited by ICP0 (9, 23), and it correlates with the formation of transcriptional activation complexes that contain IRF-3 and that bind to ISREs within ISG promoters (37). In mouse cells, this induction is dependent on IRF-3 (6). Furthermore, ICP0 is able to inhibit the IRF-3- and IRF-7-mediated activation of ISGs in transfection reporter assays (23), and ICP0 has also been implicated in the HSV-1-mediated inhibition of IRF-3 activation during Sendai virus infection (25, 26). Because ISG induction after infection with highly defective HSV-1 mutants does not require IFN, it could provide a more rapid route to the repression of viral gene expression than the stimulation of IFN synthesis followed by STAT-1-mediated signaling. Therefore, we tested whether the repression of ICP0-null mutant infection. Parallel wells were treated with IFN- at 50 U/ml and harvested as indicated. Samples were analyzed for ICP4, ISG15, and actin by Western blotting. hpi and hpt indicate hours postinfection (virus) and hours posttreatment (IFN), respectively. Mock indicates mock infected. Note that the 6-h and mock lanes for the wt infection are reversed from the expected order. The ICP4 signal in the time zero IFN sample is flow over from the adjacent lane.

5 VOL. 82, 2008 STAT-1- AND IRF-3-INDEPENDENT REPRESSION OF HSV FIG. 3. (A) Whole-cell extracts of naïve HFs and HF-BVDVN cells, which express the BVDV NPro protein, were analyzed for IRF-3 expression by Western blotting. The nonspecific band close to the 97-kDa molecular mass marker acts as a loading control. (B) The relative plaque-forming efficiencies of wt and ICP0-null mutant HSV-1 in IRF-3-depleted and naïve HFs were determined using in1863 and dl1403/cmvlacz viruses (see the legend to Fig. 2A for details on the method of calculation). Plaque numbers in HF-BVDV cells are expressed as a proportion of those in HFs. The data are the averages of six determinations, and error bars are SD. (C) Induction of ISG15 expression in HF, HF-PIV5V, and HF-BVDVN cells by IFN (20 U/ml) or in1374 infection (MOI of 10). Samples were harvested 24 h after treatment and then analyzed by Western blotting for ISG15 and actin (loading control). HSV-1 plaque formation that occurs in HFs was dependent on IRF-3. BVDV expresses a protein known as NPro that targets IRF-3 for proteasomal degradation (2, 5, 20, 42). We used a lentiviral vector to isolate transduced HFs that express BVDV NPro and are therefore highly depleted of IRF-3 (Fig. 3A). However, plaque assay experiments indicated that the plaqueforming efficiency of ICP0-null mutant HSV-1 was no greater in HF-BVDVN cells than in naïve HFs (Fig. 3B). To test whether the IRF-3-depleted cells that we generated were, as expected from the literature, unable to induce ISG expression in response to defective HSV-1, we compared the levels of expression of ISG15 following IFN treatment or in1374 infection of HF, HF-PIV5V, and HF-BVDVN cells. Virus in1374 is an HSV-1 mutant that is ICP0 null and temperature sensitive in ICP4 and has a defect in the VP16-mediated activation of IE gene expression (38). We found that both IFN treatment and in1374 infection induced ISG15 expression in naïve HFs, whereas neither one did in HF-PIV5V cells, while IFN but not in1374 induced ISG15 in HF-BVDVN cells (Fig. 3C). Although the failure of in1374 to induce ISG15 in HF-PIV5V cells was unexpected, the results indicate that HF-BVDVN cells are unable to induce ISGs in response to defective HSV-1 infection. We conclude that despite the ability of ICP0 to disrupt IRF-3-dependent events (23, 25), IRF-3 is not required for the repression of ICP0-null mutant plaque formation that occurs in HFs. The effect of IFN on wt and ICP0-null mutant HSV-1 in HFs. Although there have been a number of studies on the differential effects of IFN on wt and ICP0-null mutant HSV-1 (4, 19, 29, 30), there is limited information on the situation in HFs, the cell type in which the ICP0 defect is most pronounced. Therefore, we compared the IFN sensitivities of the wt and mutant viruses by pretreating HFs for 24 h with IFN- at various concentrations and then conducted plaque assays. For the purposes of this analysis, plaques were defined as infectious centers in which the virus had spread to surrounding cells from the initially infected cell. Plaque sizes of both the mutant and the wt virus were considerably diminished in the presence of inhibitory concentrations of IFN, with consequent (and much greater) decreases in virus yields (as described below). Figure 4A shows that wt virus plaque formation was inhibited by around 30-fold in the presence of 50 U/ml IFN-, and the sensitivity of the ICP0-null mutant was approximately fivefold greater (Fig. 4B). This finding is consistent with an independent observation that plaque formation by ICP0-null mutant HSV-1 is not massively more sensitive to IFN than that of the wt virus in HFs (3). This situation differs considerably from that in Vero cells in which wt plaque formation is only marginally inhibited by IFN pretreatment, while the mutant is at least 100-fold more sensitive (19, 29, 30). To cast light on the basis of the apparent differences between the current findings and the previous studies, we investigated the effect of IFN pretreatment on IE gene expression by wt HSV-1 in HFs. We found that IE gene expression was greatly inhibited in IFN-treated cells (Fig. 4C), a result that is entirely consistent with a previous study on the effects of IFN on HSV-1 IE gene expression in HFs (31). Therefore, the decreased differential in IFN sensitivity between wt and ICP0- null mutant HSV-1 in HFs may be a consequence of a general reduction in levels of IE gene expression, rendering the wt virus phenotypically deficient in ICP0 and other IE genes. Given the multiplicity dependence of IFN sensitivity (described below), this issue may be particularly pertinent at the low MOIs used in plaque assays. Because of differences in plaque size in the presence and absence of IFN, we also investigated the yields of virus from treated and untreated cells. This analysis gives numerically much greater inhibitory factors in the presence of IFN, because the number of infected cells in each plaque is much reduced, and it is likely that the yield of virus from individual infected cells within a plaque is also reduced. At first sight, these experiments appeared to give different conclusions from those of the plaque assays. The wt virus gave equivalent virus

6 8876 EVERETT ET AL. J. VIROL. FIG. 4. Sensitivity of wt and ICP0-null mutant HSV-1 to IFN- in HFs. (A) Cells were pretreated with the indicated amounts of IFN- (units/ml) for 24 h, and plaque assays were then conducted using threefold dilutions of in1863 and dl1403/cmvlacz. The reductions in plaque numbers in relation to untreated cells were calculated, and the mean values from several independent determinations are plotted ( SD). Where possible, plaque numbers were compared using the same virus dilution on treated or untreated cells, excluding wells with very low numbers of plaques. When it was necessary to compare plaque numbers from different virus dilutions, ratios of plaque numbers from a given virus dilution at different IFN concentration treatments were calculated and then normalized with the ratios from adjacent virus dilutions. (B) The average sensitivity of ICP0-null mutant HSV-1 compared to that of the wt was calculated for all concentrations of IFN above (A). The error bar is the SD. (C) Inhibition of wt HSV-1 IE gene expression in IFN-treated HFs. Cells were pretreated with 50 U/ml IFN- for 24 h and then infected with wt HSV-1 (strain 17 ) at an MOI of 1 PFU/cell. Samples were harvested at the indicated time points, and the levels of ICP4 and ICP0 expression were analyzed by Western blotting. A nonspecific band from a long exposure of the ICP4 blot served as a loading control. hpi, hour postinfection. yields in the presence and absence of 50 U/ml IFN- when the input virus was used at an MOI of 1.5 and 0.3, with a decrease of about 0.8 logs at an MOI of 0.1 (Fig. 5A). On the other hand, the mutant virus yields were decreased to a much greater extent, especially at an MOI of 0.1 (Fig. 5B). FIG. 5. Inhibition of wt and ICP0-null mutant HSV-1 replication in IFN-treated HFs. (A) Cells were pretreated or not with IFN- at 50 U/ml for 24 h and then infected with wt HSV-1 strain 17 at the indicated MOIs. Progeny virus was harvested 24 h after infection and titrated on U2OS cells. The mean values from two independent experiments are shown, with the error bars indicating the ranges of the two results. (B) Same as above (A) except that ICP0-null mutant dl1403 was used. The apparent differences between the data shown in Fig. 4A and 5 could be due to what might be termed effective MOI. In other words, the wt and mutant viruses were used at MOIs based on their titers in U2OS cells in order to ensure equivalent inputs on the basis of potentially active viral genomes (11, 46). However, the mutant virus initiates replication much less efficiently than the wt in HFs, with the extent of this differential dependent on MOI (11). Therefore, although the two viruses were used at the same MOI in terms of PFU in U2OS cells, the effective MOI of the mutant was far lower. Accordingly, the differences between the wt and the mutant in Fig. 5 might be explained by differences in effective MOI, especially if the degree of IFN-mediated inhibition is MOI dependent. If so, decreasing the wt MOI should increase its IFN sensitivity, while increasing the mutant MOI should decrease its sensitivity. To test this hypothesis, we investigated the effects of the MOI on IFN sensitivity in virus yield experiments. Figure 6A illustrates that wt virus yields are decreased by IFN pretreatment by almost 2 orders of magnitude at an input MOI of 0.02, and this differential is further increased as the MOI is decreased toward those used in plaque assays (of the order of an MOI of 0.001). Taken with the results shown in Fig. 5A, these data illustrate that the IFN sensitivity of wt HSV-1, when measured in terms of virus yields at 24 h after infection, is dependent on the MOI; there is a difference in yields of ap-

7 VOL. 82, 2008 STAT-1- AND IRF-3-INDEPENDENT REPRESSION OF HSV FIG. 6. Sensitivity of wt and ICP0-null mutant HSV-1 to IFN- over a range of MOIs in HFs. (A) Cells were pretreated or not with IFN- at 50 U/ml for 24 h and then infected with wt HSV-1 strain 17 at the indicated MOI. Progeny virus was harvested 24 h after infection and titrated on U2OS cells. The mean values from two independent experiments are shown, with the error bars indicating the ranges of the two results. (B) Same as above (A) except that ICP0-null mutant dl1403 was used. proximately 3 orders of magnitude between MOIs of and 0.3. Note that wt virus yields at 24 h in the absence of IFN are very similar at these two multiplicities because a 24-h infection at an MOI of is sufficient to spread throughout the cell sheet. Also consistent with the above-described hypothesis was a decrease in IFN sensitivity with increasing MOIs of the ICP0-null mutant virus (Fig. 6B). However, mutant virus yields are still depressed by IFN by 60-fold at the 24-h time point even at an MOI of 20, a value easily sufficient to give yields of mutant virus that are similar to those of the wt in the absence of IFN (Fig. 6B). In that the use of a high MOI is unable to completely overcome the inhibitory effect of pretreatment of cells with 50 U/ml IFN-, the ICP0-null mutant virus is indeed more IFN sensitive than the wt in this assay. This result may be related to the induction of ISGs seen in the mutant virus infection (Fig. 2D). However, the differential between the two viruses is highly dependent on the chosen MOI, and if both are used at low MOIs in plaque assays, their sensitivities to IFN pretreatment differ by only about fivefold (Fig. 4B). Effect of IFN on wt and ICP0-null mutant HSV-1 in PMLdepleted HFs. To summarize the data presented so far, the cell-mediated repression that targets ICP0-null mutant HSV-1 does not depend on STAT-1 signaling or IRF-3, and the apparent degree of inhibition of both wt and ICP0-null mutant FIG. 7. Virus yield experiments in HF-shLuci and HF-shPML1 cells. Cells were infected with wt HSV-1 strain 17 (A) or ICP0-null mutant dl1403 (B) at the indicated MOI, and progeny virus was then harvested 24 h after infection and titrated on U2OS cells. The mean values from two independent experiments are shown, with the error bars indicating the ranges of the two results. HSV-1 in HFs is highly dependent on the type of assay employed and the effective dose of the virus. It was suggested previously that the IFN sensitivity of ICP0-null mutant HSV-1 in mouse fibroblasts is dependent on PML (4). Expression of PML is augmented by IFN, and the protein has been implicated in mechanisms of intrinsic resistance to many virus infections (reviewed in reference 12). We investigated whether PML was required for the IFN sensitivity of ICP0-null mutant HSV-1 in HFs. The isolation of HFs that are extensively depleted of PML so that the protein is detectable by neither immunofluorescence nor Western blotting using either retroviral (15, 45) or lentiviral (13) vectors expressing an active anti-pml shrna has been documented. Here, we used HFs depleted of PML (HF-shPML1 cells) and control cells expressing an anti-luciferase shrna (HF-shLuci cells) that were generated using lentivirus transduction and characterized as described previously (13). As a preliminary to these experiments, we compared the yields of wt and ICP0-null mutant HSV-1 strains after 24 h of infection in control and PML-depleted fibroblasts at a range of multiplicities (Fig. 7). While the probability of plaque formation at a given dose of ICP0-null mutant HSV-1 is increased by about fivefold in PML-depleted HFs (15), the measurement of virus yields gives a greater differential between the two cell types, especially at a low MOI (Fig. 7). This can be explained because the mutant virus plaque numbers decrease nonlinearly with respect to virus dilution, and there comes a point when significant numbers of plaques remain on the PML-depleted cells, while the same virus dose on the control cells gives only

8 8878 EVERETT ET AL. J. VIROL. FIG. 8. Sensitivity of wt and ICP0-null mutant HSV-1 to IFN- in HF-shLuci and HF-shPML1 cells. Note the scale changes between A and B. (A) HF-shLuci cells were pretreated with the indicated amounts of IFN- (units/ml) for 24 h, and plaque assays were then conducted using threefold dilutions of in1863 and dl1403/cmvlacz. The reductions in plaque numbers in relation to untreated cells were calculated, and the mean values from several independent determinations are plotted ( SD). The method of calculation is outlined in the legend to Fig. 4A. (B) Same as above (A) except that HF-shPML1 cells were used. (C) Induction of ISG15 expression in HF-shLuci and HFshPML1 cells with IFN (20 U/ml) or in1374 infection (MOI of 10). Samples were harvested 24 h after treatment and then analyzed by Western blotting for ISG15 and actin (loading control). a very low plaque number. In the context of a virus yield experiment, this accentuates the differential between the two cell types to the point that at the lowest MOI, the yield of the mutant virus was around 2 orders of magnitude greater on the PML-depleted cells than on the controls in this experiment (Fig. 7). We then conducted a plaque assay IFN titration experiment similar to that shown in Fig. 4. The results demonstrated that ICP0-null mutant HSV-1 was approximately seven- and threefold more sensitive than the wt to IFN pretreatment (at 50 U/ml) in control and PML-depleted HFs, respectively (Fig. 8), similar to the situation in naïve HFs (a fivefold difference) (Fig. 4B). However, the wt and the mutant viruses gave approximately 4- and 10-fold more plaques, respectively, in IFNtreated HF-shPML1 cells than in the untreated cells (Fig. 8). Therefore, HFs depleted of PML appear, at face value, less able to mount an anti-hsv-1 IFN response, but this is true for both the wt and the ICP0-null mutant viruses. One interpretation of this result is that at the low MOI used in these plaque assays, IFN pretreatment results in much reduced IE gene expression (Fig. 4C) and therefore renders wt HSV-1 phenotypically deficient in ICP0 (and other IE proteins). Therefore, increased levels of wt virus plaque formation in IFN-treated PML-depleted cells may reflect the increased probability of plaque formation of the ICP0-null mutant virus in PML-depleted cells (14, 15). The implication that the depletion of PML does not inactivate an IFN response was supported by the observation that the induction of ISG15 expression by IFN treatment or in1374 infection was similar in HF-shLuci and HF-shPML1 cells (Fig. 8C). We then compared wt and ICP0-null mutant virus yields in the two cells types with or without IFN pretreatment. Cells were infected at MOIs of 1.5, 0.3, and 0.1, and the yields at 24 h after infection were then titrated. As might be expected from the results shown in Fig. 7 and 8, yields of wt HSV-1 were less sensitive to IFN in HF-shPML1 than in HF-shLuci cells, especially at the lowest MOI used (Fig. 9A and B). In addition, the yields of the mutant virus were substantially enhanced in IFNpretreated HF-shPML1 cells compared to those in HF-shLuci cells (Fig. 9C and D). The IFN sensitivity of the mutant virus in PML-depleted cells increased as the MOI was decreased. The data shown in Fig. 8 and 9 indicate that while the depletion of PML apparently renders both wt and ICP0-null mutant HSV-1 less sensitive to IFN pretreatment, it by no means protects the null mutant from IFN-mediated inhibition at a low MOI. This raises the possibility that rather than the IFN defense working directly through PML, its repressive effect may be separable from an intrinsic defense to which PML contributes by an independent mechanism. The observation that ISG15 induction is not impaired in HF-shPML1 cells (Fig. 8C) is consistent with this hypothesis. DISCUSSION This study was stimulated by previous work, using either Vero cells or mouse fibroblasts, describing the IFN sensitivity of the ICP0-null mutant HSV-1 strain and the potential role of PML in this phenomenon (4, 19, 29). Taken with reports of the ability of ICP0 to impede an IFN response (9, 25, 30), these observations suggested that the plaque-forming defect of ICP0-null mutant HSV-1 in HFs could be due to a hypersensitivity to an induced IFN response. The experiments described here set out to test this hypothesis. We stress that this paper is restricted to the hypothesis outlined above and does not impact the authenticity of previous studies of other cell types or on the potential for ICP0 to interfere with IFN response mechanisms. Our data indicate a complex situation in which the experimental outcome is hugely influenced by experimental variables such as the assay method (plaque assay or virus yield) and MOI. As these variables, particularly MOI, differentially affect the outcome of wt and

9 VOL. 82, 2008 STAT-1- AND IRF-3-INDEPENDENT REPRESSION OF HSV ICP0-null mutant HSV-1 infections, it is not straightforward to answer the deceptively simple question of whether or not ICP0-null mutant HSV-1 is markedly more sensitive to IFN than the wt virus. Our data indicate that it is important to differentiate between plaque formation per se and spread of the virus through the cell sheet. For example, wt virus plaque formation is inhibited by around 30-fold by 50 U/ml IFN- (Fig. 4A), while virus yields can be inhibited by at least 3 orders of magnitude at a low MOI (Fig. 6A). The reason for this difference is that the plaques are smaller and develop more slowly in IFNtreated cells. Thus, IFN diminishes both the probability that a cell receiving a virus particle will be able to spawn a plaque and the efficiency with which the infection spreads. This conclusion is consistent with the observation that IFN inhibits HSV-1 gene expression at multiple stages (31, 36, 41). At the level of plaque formation, perhaps the clearest data on the comparative IFN sensitivities of wt and ICP0-null mutant HSV-1 in HFs are those shown in Fig. 4A. These data show that while ICP0-null mutant HSV-1 is around fivefold more sensitive to IFN- at a low MOI, the difference is small compared to the several-hundred-fold differences seen in analogous assays of Vero cells (19, 29). When virus yields are considered, there are more robust differences between the mutant and wt viruses. Both are highly sensitive to IFN at a low MOI, and although the sensitivity decreases with the MOI in both cases, the wt becomes insensitive to IFN at an MOI of 0.3 or greater, whereas the mutant virus yields are inhibited by about 60-fold even at an MOI of 20 (Fig. 5 and 6). These data indicate that the efficiency of virus production and virus spread is inhibited by IFN more effectively in the absence of ICP0 than in its presence (Fig. 4). The issues discussed above are relevant to the questions of whether or not PML plays a role in the sensitivity of HSV-1 to IFN and whether or not this underlies the role of ICP0 in inducing the degradation of PML. It is clear that in comparison to control cells, ICP0-null mutant HSV-1 plaque formation in PML-depleted HF-shPML1 cells appears to be less sensitive to IFN by around sevenfold (Fig. 8). However, the wt virus is also less sensitive to IFN in HF-shPML1 cells, and the depletion of PML does not completely rescue the IFN sensitivity of ICP0- null mutant HSV-1. Therefore, it is evident that PML is not required for the IFN-mediated inhibition of ICP0-null mutant HSV-1 infection, as might be expected from the unimpaired IFN response of HF-shPML1 cells depicted in Fig. 8C. We propose that it is more likely that the IFN- and PML-associated repression pathways are separable and additive; thus, the plaque formation efficiency of ICP0-null mutant HSV-1 is greater in IFN-treated PML-depleted cells than in IFN-treated FIG. 9. Inhibition of wt and ICP0-null mutant HSV-1 replication in IFN-treated HF-shLuci and HF-shPML1 cells. (A) HF-shLuci cells were pretreated or not with IFN- at 50 U/ml for 24 h and then infected with wt HSV-1 strain 17 at the indicated MOIs. Progeny virus was harvested 24 h after infection and titrated on U2OS cells. The mean values from three independent experiments are shown, with the error bars indicating SD. (B) Same as above (A) except that HF-shPML1 cells were used. (C and D) Same as above (A and B) except that ICP0-null mutant dl1403 was used.

10 8880 EVERETT ET AL. J. VIROL. PML-positive cells because both repression mechanisms are active in the latter, and only one is active in the former. Given that the disruption of the IFN response pathway, by the knockout of either type I IFN receptors (22) or STAT-1 (18), hugely increases the replication and pathogenicity of ICP0-null mutant HSV-1 in mice, it is perhaps surprising that the reduction of STAT-1 to undetectable levels in HFs (Fig. 1A) does not improve the plaque formation of ICP0-null mutant HSV-1 (Fig. 2A) despite a complete elimination of the ability to respond to IFN (Fig. 1B and C and 4C). These data demonstrate without doubt that STAT-1 signaling does not underlie the defect in ICP0-null mutant plaque formation in HFs. Therefore, although ICP0-null mutant HSV-1 may be restricted more than the wt by IFN responses in vivo, it should not be assumed that the plaque-forming defect of the mutant virus in untreated HFs is due to the influence of the IFN pathway. It may also be pertinent that STAT-1 / mice will have defects in their immune system that are wide-ranging and not limited to their inability to mount an initial IFN response, and these wider defects are likely to have a significant impact on the pathogenicity of ICP0-null mutant HSV-1. The above-described considerations are also relevant to the lack of involvement of IRF-3 in the plaque-forming defect of ICP0-null mutant HSV-1 in HFs (Fig. 3). Highly defective HSV-1 mutants that lack ICP0 and other major viral transactivators induce an IFN-like response involving the formation of complexes containing IRF-3 on ISREs in the promoters of ISGs (9, 28, 32, 37). This cellular response is disarmed by wt HSV-1, and ICP0 has been implicated in this viral evasion strategy (9, 23, 25, 30). Furthermore, ICP0 can disrupt IRF-3- dependent promoter activation in transfection reporter assays (23). Our observation that ISG15 expression is induced during infection by an ICP0-null mutant but not by the wt (Fig. 2D) is consistent with those previously published findings. Therefore, it is a surprise that the depletion of IRF-3 does not improve plaque formation by ICP0-null HSV-1. However, the previous studies used MOIs of 5 (32) or greater (9, 28) and detected the induction of ISGs at 6 h (32) or 24 h or more (9, 28) after infection. In one study, the induction of ISGs was observed only at MOIs greater than 10 and at times greater than 24 h after infection (9). It is possible that the induction of ISGs during ICP0-null mutant HSV-1 infection (Fig. 2D) is too slow to inhibit the initial steps of commitment of a cell to the lytic cycle and, thus, plaque formation. Accordingly, the failure of IRF-3 depletion to complement the plaque formation of an ICP0-null mutant virus suggests that IRF-3-dependent ISG induction does not underlie this particular defect of these viruses. Nonetheless, it is likely that, just as in the case of STAT-1, IRF-3 plays an important role in controlling HSV-1 infections in vivo. Taken together, our data suggest that the plaque-forming defect of ICP0-null mutant HSV-1 in HFs cannot be explained solely by an induced IFN pathway response. Such a mechanism would require the activation of signaling pathways and the synthesis of antiviral proteins before any inhibitory effect on the virus could be mounted. This may be too slow to substantially impede even ICP0-null mutant HSV-1 in the initial stages of infection. Accordingly, it appears more likely that the efficient inhibition of ICP0-null mutant gene expression, which underlies the plaque-forming defect, is due to an intrinsic defense mechanism that comprises preexisting inhibitory molecules. ACKNOWLEDGMENTS The work in Glasgow was supported by the Medical Research Council and in part by the European Commission Framework 6 SME- STREP project FP , TargetHerpes. 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