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- MICROBIOLOGY 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 herpesvi- rus, 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. www.pnas.org/cgi/doi/10.1073/pnas.1114232109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 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 un- infected 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.