(STAT3) and Survivin Induction by Varicella-Zoster Virus Promote Replication and Skin Pathogenesis

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Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis

Nandini Sena,1, Xibing Chea, Jaya Rajamania, Leigh Zerbonia, Phillip Sunga, Jason Ptacekb, and Ann M. Arvina

aDepartments of Pediatrics and Microbiology and Immunology and bCenter for Clinical Sciences Research, Stanford University School of Medicine, Stanford, CA 94305

Edited by Bernard Roizman, University of Chicago, Chicago, IL, and approved November 29, 2011 (received for review August 30, 2011) Varicella-zoster virus (VZV) is a human α-herpesvirus that causes cellular kinases, including the Jak and Src family kinases and varicella (chickenpox) during primary infection and zoster (shin- growth factor receptor tyrosine kinases (11). STAT3 activation gles) upon reactivation. Like other viruses, VZV must subvert the leads to overexpression of genes involved in tumorigenesis and is intrinsic antiviral defenses of differentiated human cells to pro- constitutive in most primary human tumors (11–13). STAT3 duce progeny virions. Accordingly, VZV inhibits the activation plays a significant role in the pathogenesis of the γ-herpesviruses, of the cellular transcription factors IFN regulatory factor 3 (IRF3) Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein–Barr and signal transducers and activators of transcription 1 (STAT1), virus (EBV), and herpesvirus saimiri (14–16), all of which exploit thereby downregulating antiviral factors, including IFNs. Con- the oncogenic effects of phosphorylated STAT3 (pSTAT3). versely, in this study, we found that VZV triggers STAT3 phosphor- Transcription mediated by pSTAT3 controls several apoptotic ylation in cells infected in vitro and in human skin xenografts in pathway genes, including the Bcl family and the inhibitors of SCID mice in vivo and that STAT3 activation induces the anti-apo- apoptosis (IAP) family genes. Survivin, an inducible member of ptotic protein survivin. Small-molecule inhibitors of STAT3 phos- the IAP family, is abundant in cancers and tissues that contain phorylation and survivin restrict VZV replication in vitro, and VZV proliferating cells (17, 18). STAT3 binds to the survivin pro- infection of skin xenografts in vivo is markedly impaired by the moter, and inhibition of STAT3 signaling reduces survivin ex- administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and pression (19, 20). In these experiments, we found that survivin survivin are required for malignant transformation caused by mediates a necessary virus-enhancing effect of STAT3 activation γ-herpesviruses, such as Kaposi’s sarcoma virus. We show that on VZV, a lytic herpesvirus. STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 ac- Results tivation is critical for the life cycle of the virus because VZV skin VZV Directs STAT3 Phosphorylation in Infected Cells Independently of infection is necessary for viral transmission and persistence in the Secreted Factors. pSTAT3 expression measured by mean fluo- human population. Therefore, we conclude that takeover of this rescence intensity (MFI) was reproducibly approximately two- major cell-signaling pathway is necessary, independent of cell fold higher in human embryonic lung fibroblasts (HELF) transformation, for herpesvirus pathogenesis and that STAT3 infected with VZV-GFP virus compared with uninfected HELF activation and up-regulation of survivin is a common mechanism from the same monolayer or control uninfected (UI) HELF (Fig. important for the pathogenesis of lytic as well as tumorigenic 1A and Fig. S1 A and B). pSTAT3 was comparable in VZV+ herpesviruses. HELF and in IFNα-treated, uninfected HELF, and expression in VZV− HELF was similar to uninfected HELF. When equal he life cycle of varicella-zoster virus (VZV) in the human numbers of sorted VZV+ and VZV− populations were analyzed Thost depends on its tropism for T cells, skin, and neurons for pSTAT3 by immunoblot, pSTAT3 expression was enhanced within sensory ganglia (1). As shown in the SCID mouse model in the VZV+ HELF, and total STAT3 levels were similar in of VZV pathogenesis, infected human T cells transport the virus both populations (Fig. 1B). VZV glycoprotein E (gE) expression to epidermal cells in human skin xenografts and to neural cells in confirmed the efficiency of cell separation. When pSTAT3 ex- dorsal root ganglia xenografts (2, 3). VZV establishes latency in pression was analyzed in human tonsil T cells, VZV+ CD3+ T sensory ganglia; upon reactivation, the virus migrates to the skin cells exhibited ∼1.5-fold higher pSTAT3 compared with control via axonal transport to cause zoster. T cells cocultured with uninfected HELF (Fig. S1C). In contrast, VZV modulates several signaling pathways to replicate effi- pSTAT3 in VZV− CD3+ T cells was similar to mock-infected T ciently in vitro, and these regulatory effects are especially cells. Thus, STAT3 activation was induced in both HELF and T important in differentiated skin cells infected in vivo. VZV cells and only in the VZV-infected cell populations in vitro. interferes with IFN induction and signaling via inhibition of IFN To determine whether VZV-infected cells retained respon- regulatory factor 3 (IRF3), NFκB, and STAT1 in vitro and in siveness to exogenous STAT3 activation, VZV-infected HELF skin (4, 5). However, the pathogenesis of VZV skin infection were treated with IFNα for 30 min before phosphoflow analysis. requires a mechanism to overcome the constitutive IFNα ex- Both VZV+ and VZV− cells responded to IFNα with a shift in pression by epidermal cells that accounts for the 10- to 21-d in- MFI (Fig. 1C), but pSTAT3 expression was higher in VZV+ cells terval between VZV transfer into skin and the appearance of exposed to IFNα than in VZV− cells. Thus, although VZV lesions at skin surfaces (6). How VZV overcomes this cutaneous IFN barrier and produces skin vesicles is not known. The STATs are ubiquitous transcription factors with many Author contributions: N.S. and A.M.A. designed research; N.S., X.C., J.R., L.Z., P.S., and J.P. cellular functions and are at the junction of several cytokine- performed research; N.S. and A.M.A. analyzed data; and N.S. and A.M.A. wrote the paper. signaling pathways (7, 8). Of the seven STAT family proteins, The authors declare no conflict of interest. STAT3 exerts widespread effects through transcriptional up- This article is a PNAS Direct Submission. regulation of genes encoding proteins involved in cell survival, 1To whom correspondence should be addressed. E-mail: [email protected]. cell cycle progression, and homeostasis (9, 10). STAT3 is acti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vated by phosphorylation via several receptor and nonreceptor 1073/pnas.1114232109/-/DCSupplemental.

600–605 | PNAS | January 10, 2012 | vol. 109 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1114232109 Downloaded by guest on September 23, 2021 Fig. 1. VZV infection directs STAT3 phosphorylation in human ﬁbroblasts independently of secreted factors. (A) Infected and uninfected HELF were analyzed at 24 hpi by phosphoﬂow for pSTAT3 expression. The MFI of pSTAT3 expression from different experiments was plotted and represented as a bar chart; uninfected HELF (UI) and HELF treated with IFNα were the negative and positive controls, respectively. (B) Total and pSTAT3 expression in FACS-sorted GFP+ and GFP− populations. (C) FACS analysis for detection of pSTAT3 expression in VZV-GFP–infected cells treated or untreated with IFNα.(D) Conditioned media from uninfected and VZV-infected HELF were tested for the presence of secreted cytokines by Luminex assay at 24 hpi. (E) Culture supernatants from uninfected and VZV- infected HELF (24 hpi) were added to fresh monolayers of HELF. Lysates from untreated control HELF (“C”), HELF treated with OSM, HELF treated with two increasing volumes of supernatant from uninfected HELF (UI), and VZV-infected HELF (VZV) were probed for pSTAT3 expression by immunoblotting.

robustly inhibits IFN induction (4), the ability of exogenous IFNα VZV infection of human skin xenografts in vivo. Skin xenografts to trigger STAT3 phosphorylation in infected cells is unimpaired. in two groups of five mice (two xenografts/mouse) were in- To further substantiate that STAT3 activation was not a sec- oculated with a VZV recombinant expressing firefly luciferase ondary effect, supernatants collected 24 h after VZV or mock (22). Group I received DMSO and group II was treated with S3I- infection of HELF were tested using a 51-plex human cytokine 201 every other day from day 0–16 after VZV inoculation, and D array (Fig. 1 ), which included interleukin 6 (IL6) and leukemia all were imaged daily for 21 d (Fig. 3). Xenografts were not inhibitory factor (LIF), which are known to trigger pSTAT3. Se- infected in one S3I-201 animal (#8); the inhibitor did not affect creted IL6 was reduced whereas LIF showed a slight 1.4-fold in- skin cell morphology or total STAT3 (Fig. S4). α β crease in supernatant from VZV-infected HELF; IFN and IFN By day 10, 5/10 (50%) xenografts in the DMSO group showed controls were not detected. Conditioned media from uninfected bioluminescence compared with only 1/8 (12.5%) in the S3I-201 and VZV-infected HELF were also evaluated to determine group (P = 0.028 at the 5% level; two-sample Z test to compare whether pSTAT3 might result from secreted factors below de- proportion of positives) (Fig. 3). On day 12, the number of tection or not included in the multiplex assay and if the minor positive xenografts increased to 6/10 (60%) in the DMSO group increase in LIF secretion was biologically relevant. Supernatants recovered at 24 h were added to HELF for 24 h; oncostatin M vs. 3/8 (38%) in the S3I-201 group. By day 15, the S3I-201 group (OSM) was a positive control. When lysates were tested for continued to have fewer positive xenografts compared with the P pSTAT3 by immunoblot, media containing OSM induced DMSO group (1/8 vs. 7/10; = 0.001), and signal intensity was pSTAT3, but conditioned media from VZV-infected and un- lower in those that were positive. infected HELF did not (Fig. 1E). Thus, secreted factors released S3I-201 treatment reduced VZV replication on the basis of from VZV-infected cells, including LIF, were unlikely to consti- the bioluminescence signal and the number of positive xeno- tute a major mechanism for pSTAT3 up-regulation during VZV grafts compared with DMSO-treated mice throughout the 21-d replication. The data also confirmed that STAT3 was phosphor- period (Fig. 4). The kinetics of VZV replication in DMSO- ylated in the infected but not in the uninfected cell population. treated animals peaked at day 19–21, as expected from prior studies (23). The biological activity of the inhibitor was con- Inhibition of STAT3 Phosphorylation Restricts VZV Replication and firmed because withdrawing the drug resulted in increased VZV Cell–Cell Spread in Vitro. S3I-201, an amidosalicylic compound, replication in 3/8 xenografts in the S3I-201 group (Fig. 4). At inhibits STAT3 phosphorylation and dimerization, thereby 21 d, VZV was recovered from 6/7 bioluminescence-positive blocking its transactivating function (21). VZV spread was re- xenografts in the DMSO group (titer: 0.3 × 102 – 2 × 104 pfu) fi stricted signi cantly in the presence of S3I-201 compared with compared with only 1 (titer: 1.4 × 103) in the S3I-201 group. DMSO (Fig. 2A) whereas no effect was observed in untreated and VZV DNA was detected by PCR in all bioluminescence-positive MICROBIOLOGY DMSO-treated HELF (Fig. S2). Inhibitor treated and untreated xenografts and in none of those that were negative. Thus, VZV cells did not differ in cell morphology, viability, or number. The infection was typically blocked completely in the S31-201 group. mean VZV titer was ∼1.5-fold less in HELF infected in the pres- These experiments indicated that STAT3 phosphorylation was ence of S3I-201 compared with DMSO at 48 hours post infection required for efficient VZV replication and spread in human skin (hpi) (Fig. 2B). Inhibition of pSTAT3 had more pronounced effects when the inoculum titer was lower (Fig. 2C). With an in- in vivo. Notably, blocking STAT3 activation had a much more oculum of 0.8 × 103 pfu, the difference in viral titers between substantial effect on VZV infection in vivo than could be de- treated and control HELF was ∼1.5-fold lower compared with tected in cultured cells in vitro. <0.5 with an inoculum of 8.0 × 103 pfu. S3I-201 treatment also reduced VZV plaque size significantly in the titration plates VZV Infection of Skin Is Associated with STAT3 Phosphorylation in (Fig. 2D). Furthermore, pSTAT3 inhibition reduced VZV in- Vivo. When skin sections were analyzed for pSTAT3 and IE63, fectionoftonsilTcells;50%fewerTcellswereinfectedinthe an abundant VZV protein, pSTAT3 expression was unique to presence of S3I-201 compared with DMSO (Fig. S3). the infected skin tissues and was undetectable in sham inoculated xenografts (Fig. 5A). In skin, the populations of cells that Role of STAT3 Phosphorylation in the Pathogenesis of VZV Skin expressed pSTAT3 and those that showed IE63 expression did Infection. To establish biological relevance, we investigated the not overlap completely; pSTAT3 was also detected in cells sur- consequences of interfering with STAT3 phosphorylation on rounding the foci of VZV-infected cells.

Sen et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | 601 Downloaded by guest on September 23, 2021 STAT3 is itself such a target gene, STAT3 transcript levels were unchanged in VZV-infected HELF (Fig. 6A), consistent with no change in total STAT3 protein (Fig. 1B and Fig. S5A). To identify other downstream targets of pSTAT3 that are poten- tially regulated during VZV infection, the transcription of LIF, cyclin D1, survivin, and Bcl-xl genes was evaluated (Table S1). At 24 h after VZV infection, survivin transcripts increased in VZV- infected compared with uninfected HELF by RT-PCR (∼2.8- fold) whereas transcription of Bcl-xl and cyclin D1 genes remained largely unchanged (Fig. 6A). LIF gene transcripts increased slightly (1.3-fold), consistent with the low level of LIF protein secretion (Fig. 1E). When confirmed by quantitative PCR, survivin gene transcription increased by 16-fold in VZV- infected HELF compared with uninfected cells, which was comparable to the 15-fold increase observed in IL6-treated HELF (Fig. S5B). Survivin gene up-regulation was further evaluated in HELF that were inoculated with VZV-GFP, sorted into VZV+ and VZV− cell populations after 24 h, and analyzed by RT-PCR. The up-regulation of survivin gene transcription occurred pre- dominantly within VZV-infected cells whereas cyclin D1 gene transcripts were only marginally increased compared with uninfected cells (Fig. 6B). Survivin gene transcription was reduced when VZV-infected HELF were treated with the pSTAT3 in- C Fig. 2. Inhibition of STAT3 phosphorylation restricts VZV replication and hibitor S3I-201 compared with DMSO (Fig. 6 ). Furthermore, spread in vitro. (A) VZV-GFP–infected HELF were treated with DMSO or S3I- survivin gene transcription was associated with induction of 201; virus spread was determined at 48 hpi by fluorescence microscopy, and survivin protein. Survivin expression was enhanced in VZV- nuclei were stained with Hoechst. (B) Infectious virus yields from HELF infected infected cell lysates compared with the uninfected lysate (lane with VZV-GFP in the presence of S3I-201 or DMSO were determined at 48 hpi 3 vs. lane 1 in Fig. 6D) and induction of survivin protein was by titration on melanoma cells; plaques were detected by staining with blocked by the pSTAT3 inhibitor (lane 4 in Fig. 6D). No deg- human polyclonal anti-VZV IgG. Plaques from representative wells are radation of IκBα was observed compared with the uninfected shown; graph shows mean titers ± SEM in the presence and absence of the κ – control, indicating that survivin increase is unlikely to be NF B- pSTAT3 inhibitor. (C) DMSO- and S3I-201 treated HELF were inoculated with κ increasing titers of VZV, and replication was assessed at 48 hpi. (D) Plaque dependent and that VZV is known to inhibit NF B (5). sizes in melanoma cells infected with VZV propagated in DMSO- and S3I- We next determined if survivin expression was also up-regu- 201–treated HELF for 48 h were determined using ImageJ; graph shows the lated during VZV infection of skin in vivo. As expected, VZV mean size ± SEM of 10 plaques from each condition. glycoprotein E (red) was expressed prominently on infected cell membranes (Fig. 6E). Survivin expression (green) was detected in the nuclei and cytoplasm of gE-expressing cells (Fig. 6E and To further verify pSTAT3 induction, skin sections were Fig. S5C). Some nearby cells that did not express gE also con- B stained for ORF61 protein and pSTAT3 (Fig. 5 ). ORF61 is tained survivin (Fig. S5D). This pattern of survivin expression expressed in infected cell nuclei very early after infection (24). was as observed for pSTAT3, which was found in VZV-infected ORF61-positive cells (green) that also expressed pSTAT3 (red) cells and in cells in close proximity to infected skin cells. were observed. However, cells immediately adjacent to VZV lesions that had no detectable ORF61 expressed pSTAT3, con- Inhibition of Survivin Restricts VZV Replication and Spread in Vitro. sistent with results using IE63 to identify VZV-infected skin Given these findings, YM155, a small-molecule inhibitor of cells. These observations suggest that STAT3 activation may be survivin transcription, was used to assess whether increased induced before expression of viral proteins at detectable levels. survivin expression was required for VZV replication and spread (25). VZV titers decreased in the presence of YM155- compared VZV Infection Leads to Up-Regulation of Survivin Expression in Vitro with DMSO-treated HELF at 48 hpi (Fig. 7A). VZV spread in and in Vivo. We next determined if enhanced STAT3 phosphor- YM155-treated HELF was also much more restricted compared ylation had functional effects in VZV-infected cells by assessing with DMSO-treated HELF (Fig. 7B); mean plaque sizes were expression of target genes regulated by pSTAT3. Although ∼0.2 mm2 with YM155 compared with ∼0.4 mm2 with DMSO.

Fig. 3. Assessment of the effects of inhibiting STAT3 phosphorylation on VZV replication in skin xenografts in vivo. Human skin xenografts in SCID mice were infected with VZV expressing ﬁreﬂy luciferase. Mice, with two xenografts each, were treated with DMSO (group I: mouse #1–5) or S3I-201 (group II: mouse #6–10) beginning on the day of inoculation through day 16. Xenografts in mouse #8 were left uninfected to assess the effects of S3I-201 treatment on human skin. VZV replication in the xenografts was monitored by bioluminescence imaging for 21 d; images shown were taken on the days post infection as indicated.

602 | www.pnas.org/cgi/doi/10.1073/pnas.1114232109 Sen et al. Downloaded by guest on September 23, 2021 and inhibiting STAT3 activation was associated with markedly diminished T-cell infection. The pathogenic potential of VZV depends on infection of T cells, which transfer the virus to skin. Thus, VZV regulation of STAT3 is important in virus tropism for T cells as well as skin cells, both of which are essential targets for VZV’s life cycle in the human host. Constitutive expression of pSTAT3 and survivin is a well- known characteristic of tumor cells and cells transformed by oncogenic viruses, including KSHV and EBV (14, 26–28). Our study of VZV shows that STAT3 activation and survivin expression also support lytic herpesvirus replication not only in vitro but also in differentiated human skin cells within their tissue microenvironment in vivo. Most oncogenic viruses induce a persistent activation of STAT3. In contrast, the process of α-herpesvirus takeover of the host cell is directed toward as- sembly and release of large quantities of infectious virus particles and requires only short-term preservation of the infected cell. VZV has no mechanism to abort its lytic program in skin or T cells. Thus, these experiments indicate that STAT3 activation need not be prolonged or support oncogenesis to have important Fig. 4. Inhibition of STAT3 phosphorylation restricts VZV replication in skin proviral effects for herpesviruses. in vivo. The average bioluminescence from the skin xenografts that had Investigating STAT3 regulation by VZV has the advantage that a positive signal (y axis) was plotted against the days post infection (x axis) in consequences for pathogenesis can be established in the SCID the DMSO- and S3I-201–treated mice (Upper). Arrows indicate discontinua- mouse model in vivo. VZV interactions with differentiated human tion of DMSO and S3I-201 treatment on day 16. (Lower) The number of skin cells within tissues can be examined directly without modulation xenografts with a positive bioluminescence signal on each day. by adaptive immunity and signaling pathways involved in host cell defense, and changes in cell signaling elicited by VZV replication can be assessed functionally by treating animals with monoclonal Impaired VZV replication in the absence of survivin was further antibodies or small-molecule inhibitors in vivo (6). In these experi- confirmed by decreased expression of VZV proteins IE62, ments, inhibition of STAT3 by i.p. administration of S3I-201 dra- ORF4, and ORF11 at 24 hpi in the presence of increasing matically reduced the capacity of VZV to establish or maintain concentrations of the survivin inhibitor (Fig. 7C). infection in human skin xenografts. As reported in studies of S3I- 201 in tumor models (29), mice given the drug had no adverse Discussion effects, and skin xenografts showed no drug-related changes. Our study demonstrates that VZV activation of STAT3 is a crit- Therefore, the refractoriness to VZV infection can be attributed ical determinant of the pathogenesis of skin infection in vivo. to interference with STAT3 phosphorylation. Furthermore, VZV-induced activation of STAT3 resulted in the The importance of epidermal cell production of type I IFNs expression of survivin in virus-infected cells, and survivin was in innate control of VZV was demonstrated by extensive lesion necessary for optimal VZV replication. Interference either with formation when anti-IFNα/β antibody was given to SCID mice STAT3 phosphorylation or directly with survivin using small- with infected skin xenografts (6). Although STAT1 and STAT3 molecule inhibitors impaired the cell–cell spread of VZV and activation can be synergistic, some studies show that these pro- diminished or blocked the production of infectious virus progeny. teins may be antagonistic (30). pSTAT1 is not detected in VZV- VZV also up-regulated pSTAT3 in primary human tonsil T cells, infected epidermal cells whereas nuclear pSTAT1 is prominent in the uninfected cells (6). Conversely, STAT3 activation in VZV-infected epidermal cells and impaired skin infection when STAT3 phosphorylation is inhibited now identify a mechanism by which VZV can overcome this substantial IFN barrier in skin. Interestingly, pSTAT3 nuclear expression was detected in some epidermal cells that did not express VZV proteins but were in close proximity to pSTAT3-positive infected cells, suggesting that these cells may be in an early stage of infection. Because IFNα is present in VZV-infected skin tissues, it is also of interest MICROBIOLOGY that exogenous stimulation of VZV-infected HELF with IFNα had an additive effect on pSTAT3 expression. Once STAT3 activation has occurred in VZV-infected skin cells, local IFNα might contribute to the change in the pSTAT expression pattern. Thus, VZV-mediated STAT3 phosphorylation in epidermal cells may suppress antiviral responses mediated by pSTAT1 through a negative feedback signaling loop, which permits cell–cell spread and eventually produces vesicles containing infectious Fig. 5. STAT3 phosphorylation in VZV-infected skin in vivo. (A) VZV-infected virions at the skin surface. and uninfected skin sections obtained 21 days post inoculation were ana- Although STAT3 activation has varied effects, survivin ex- lyzed by immunohistochemistry. Sections were probed with antibodies to pression is a well-known consequence (31). Survivin was the only VZV-IE63 (to identify foci of infected cells) and pSTAT3; treatment with calf fi intestinal phosphatase confirmed the specificity of pSTAT3 detection. (B)For one of ve STAT3-responsive genes, including Bcl-xl, another immunofluorescence, VZV-infected skin sections were stained with anti- anti-apoptotic factor, induced in VZV-infected cells. Inhibiting bodies to the viral protein ORF61 (Alexa Fluor 488; green) and pSTAT3 (Alexa survivin with YM155 had effects similar to blocking STAT3 ac- Fluor 594; red) and examined by confocal microscopy. Nuclei were stained tivation. It is also possible that nuclear survivin observed in some with Hoechst. VZV-infected cells modulates the cell cycle to benefit viral

Sen et al. PNAS | January 10, 2012 | vol. 109 | no. 2 | 603 Downloaded by guest on September 23, 2021 Survivin also modulates cell proliferation; human cytomegalovirus was recently shown to mediate endothelial cell proliferation and angiogenesis associated with survivin expression (34, 35). However, we observed no difference in expression of the Ki67 proliferation marker in VZV-infected and uninfected skin xenografts (36). From these observations, we propose that the anti- apoptotic activity of survivin is of primary importance for VZV skin pathogenesis by maintaining the VZV-infected cell long enough to allow sufficient production and transfer of infectious virus particles to neighboring epidermal cells. Interestingly, survivin is expressed in human keratinocytes that compose the outer root sheath of hair follicles (37). Because these are the first cells that express VZV proteins after T-cell transfer of the virus, VZV may take advantage of the proviral function of this endogenous survivin to support the initial phase of skin infection. Whereas the oncogenic γ-herpesviruses have mechanisms that induce survivin directly, our experiments indicate that survivin expression in VZV-infected cells is secondary to STAT3 activation although a direct mechanism is not excluded. Survivin is stimu- lated by KSHV through the binding of latency-associated nuclear antigen (LANA) to the survivin promoter and by EBV latent Fig 6. VZV infection leads to up-regulation of survivin transcription and membrane protein 1 (LMP1) via the NFκB and AP1 pathways (27, translation in vitro and in vivo. (A) Assessment of indicated pSTAT3-reg- 28). Both LANA and LMP1 also induce pSTAT3 but have not ulated cellular transcripts by RT-PCR in uninfected and VZV-infected HELF been linked to survivin induction by either of these two γ-herpes- at 24 hpi. (B)VZV-GFP–infected HELF were flow-sorted to recover GFP+ virus proteins. Survivin up-regulation in VZV-infected cells did − and GFP populations. RNA from each sorted cell population was pro- not correlate with NFκB activation, and VZV blocks NFκB acti- cessed for detection of indicated transcripts. (C) Detection of survivin vation in vitro and in vivo, indicating that this pathway is not an transcripts in HELF infected with VZV in the presence of DMSO and S3I-201. (D) Immunoblot detection of survivin, pSTAT3, and IκBα expression from alternative to survivin induction through pSTAT3 (5). pSTAT3 in untreated, uninfected HELF (lane 1), IL6-treated HELF (lane 2), VZV-infec- virus-infected cells has been associated with interactions between ted HELF + DMSO (lane 3), and VZV-infected HELF + S3I-201 (lane 4). (E) various host cell kinases and viral factors. A role for cellular VZV-infected and uninfected skin sections were stained with antibodies to kinases in STAT3 activation in VZV-infected cells is suggested by the viral glycoprotein gE (Alexa Fluor 594; red) and survivin (Alexa Fluor the observation that resveratrol, which inhibits pSTAT3 mediated 488; green) and examined by confocal microscopy. Nuclei were stained by cellular kinases, has antiviral activity against VZV (38, 39). The with Hoechst. role of cellular and viral kinases in pSTAT3 and survivin up-regulation during VZV infection warrants investigation. replication. Nuclear survivin has been associated with virus-me- In summary, these observations document the activation of diated induction of the S phase of the cell cycle that supports STAT3 by VZV and its importance for herpesvirus replication not adenovirus replication (32), and an increase in progression to the only in vitro but also in vivo. We suggest that pSTAT3 antagonism S/G2 phase has been observed in VZV-infected cells (33). of pSTAT1 facilitates spread of the virus in skin despite the

Fig. 7. Inhibition of survivin expression restricts VZV replication and spread in vitro. (A) HELF were infected with VZV-GFP in the presence of YM155 or DMSO, and infectious virus yields were determined at 48 hpi by titration on melanoma cells. (B) VZV-GFP–infected HELF were treated with DMSO or YM155; plaques were detected by staining with anti-VZV IgG at 48 hpi. Plaque sizes were determined using ImageJ; the graph shows the mean size ± SEM of 30 plaques. (C)Expres- sion of VZV proteins was assessed in VZV-infected HELF lysates (24 hpi) treated with YM155 or DMSO.

604 | www.pnas.org/cgi/doi/10.1073/pnas.1114232109 Sen et al. Downloaded by guest on September 23, 2021 presence of IFNα and, in addition, that VZV-induced STAT3 In Vivo Imaging. Xenografts prepared from human fetal tissues (obtained activation enhances infection via survivin expression and inhibition from Advanced Bioscience Resources with informed consent according to of apoptosis (Fig. S6). Identifying host and viral proteins involved federal and state regulations) were engrafted in male homozygous C.B-17 in the control of this complex STAT3-signaling pathway is of in- scid mice and injected with VZV-infected HELF at 6 wks; mice were injected terest because its biological relevance for pathogenesis is estab- i.p. with S3I-201 or DMSO every other day from day 0–16 (29). lished. From this work, we conclude that STAT3 activation and Bioluminescence was measured from day 0–21 in mice given 3 mg D-lu- the induction of survivin is a common mechanism necessary for ciferin (Caliper Life Sciences) and imaged after 5 min (Xenogen IVIS 200). the pathogenesis of lytic as well as tumorigenic herpesviruses. Luminescence values were calculated with Living Image software-2.50.1. Materials and Methods RNA Isolation and RT-PCR Assay. Total RNA isolated from infected and un- Viruses. ORF10-GFP (VZV-GFP; gift from T. C. Heineman, GlaxoSmithKline infected HELF (TRIzol Reagent; Invitrogen) was reverse-transcribed with ﬁ ﬁ Biologicals) and rOkaF62/63RL-expressing luciferase (22) were used for a rst-strand cDNA synthesis kit (Invitrogen); 5% of the rst-strand reaction in vitro and in vivo studies, respectively. was used for RT-PCR to detect survivin, Bcl-xl, STAT3, LIF, cyclin D1, and GAPDH using primers listed in Table S1 (40). HELF infected with VZV-GFP for Reagents. S3I-201 (Calbiochem) was used at 100 μM for 48 h in vitro and 24 h were sorted into GFP+ and GFP− populations using Vantoo sorter. An 5 mg/kg for in vivo i.p. administration into the xenotransplanted mice (29). equal number of cells from each population were used for cDNA synthesis YM155 (Selleck Chemical) was used at 0.25 or 0.5 nM for 9 h. OSM (0.5 (SuperscriptIII Cell Direct cDNA Synthesis System; Invitrogen) and RT-PCR.

µg/mL; Biovision), IFNα (104 U/mL; Sigma), and IL6 (100 ng/mL; Cell Signaling Technology) were used to induce pSTAT3. Luminex Assay. Secreted cytokines were detected using the Luminex 200 IS System. Flow Cytometry. Single-cell suspensions of VZV-GFP–infected HELF were ﬁ made in FACs buffer, xed in paraformaldehyde (1.5%), permeabilized in ACKNOWLEDGMENTS. We thank Dr. Adrish Sen for helpful discussions and methanol, stained using CD3-PE and Alexa Fluor 647-pSTAT3-Y705, and Tim Knaak and Yael Rosenberg for technical assistance. This work was analyzed using BD Biosciences LSR II. supported by NIH Grants AI20459 and AI053846.

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