Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms

Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses

Inhibition of IFN-induced JAK-STAT signaling by VZV.

<p>(A) MRC5 cells were infected with VZV.eGFP (ratio 5:1) for 48 hours and stimulated for 20 minutes with 5000 U/ml uIFN. The lysates were analyzed for expression of STAT1, phosphorylated STAT1, STAT2, phosphorylated STAT2 (pSTAT2) and IRF9 using SDS-PAGE and western blot with specific antibodies. Lysates were stained for ORF31 and ORF63 to confirm viral infection and GAPDH was used as a loading control. (B/C) HEK 293T cells were co-transfected with plasmids encoding ISRE-luciferase and the indicated viral proteins. At 42 hours post transfection, the cells were stimulated with uIFN for 6 hours after which luciferase expression was measured. At this time part of the cells were lysed and expression of the transfected proteins was verified by SDS-PAGE and western blot using FLAG- and ORF63-specific antibodies. GAPDH was used as a loading control. (D) MRC5 cells and TRFs were co-infected with VZV AdORF63 at an MOI of 20, and AdTA at an MOI 10. The infected cells were incubated with the indicated concentrations of Dox. At 48 hpi the cells were stimulated with 5000 U/ml uIFN for 20 minutes. The lysates were analyzed for expression of STAT2, phosphorylated STAT2 (pSTAT2), IRF9, ORF63, and GAPDH by SDS-PAGE and western blot using specific antibodies.</p

SVV ORF63 inhibits IFN-stimulated gene expression.

<p>(A) TRFs were co-infected with AdTA at MOI 10 and AdORF63 at MOI 20 in the presence of 1000 ng/ml doxycycline (Dox) to suppress ORF63 expression or 1 ng/ml Dox to allow for ORF63 expression. At 26, 34, 38 and 40 hours p.i. the cells were stimulated with 5000 U/ml uIFN for 16, 8, 4 and 2 hours, respectively, after which RNA was harvested to quantify Mx-1 and ISG54 mRNA expression by qPCR. ORF63 mRNA expression was confirmed by qPCR using ORF63-specific primers. Data were normalized to the level of GAPDH mRNA expression in each sample and are shown as fold induction relative to unstimulated control cells. Shown are the means ± standard error of the mean of two independent experiments with three replicates/sample in each experiment. (B) TRFs were mock-infected or co-transduced with AdORF63 (MOI 20) and AdTA (MOI 10) and cultured in the presence of 1000 ng/ml or 1 ng/ml Dox. 40 hours p.i. the cells were incubated with 5000 U/ml uIFN for 8 hours and lysed in SDS sample buffer. Lysates were analyzed for ISG15, ISG54 and Mx-1 expression by SDS-PAGE and western blot. ORF63 staining confirmed expression of the protein in cells treated with 1 ng/ml Dox. GAPDH was used as a loading control. One representative experiment out of three independent experiments is shown.</p

ORF63 does not affect STAT2 activation but promotes IRF9-degradation.

<p>(A) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 in the presence of 1000 ng/ml Dox (control) or 1 ng/ml Dox for 48 hours. The cells were stimulated with 5000 U/ml uIFN for 20 min and lysates were analyzed via SDS PAGE and western blot for steady expression and phosphorylation status (p) of the indicated members of the JAK-STAT pathway. GAPDH was used as a loading control. ORF63 expression was confirmed using a specific antibody. One representative experiment out of three independent experiments is shown. The GAPDH signal was used a loading control. (B) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 and simultaneously incubated with decreasing amounts of Dox. 48 hours p.i. the cells were stimulated with 5000 U/ml uIFN for 20 minutes, lysed and stained for IRF9, ORF63 and GAPDH. (C) Graph showing IRF9 and ORF63 expression normalized to GAPDH. Shown are the means ± standard error of two independent experiments. *: p-value < 0.005, **: p-value < 0.0005, ***: p-value < 0.00005; compared to expression in cells incubated with 1000 ng/ml Dox. (D) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 and incubated with decreasing amounts of Dox. 48 hours p.i. the cells were lysed and stained for STAT1, STAT2, ORF63 and GAPDH. (E) Graphs showing STAT1 and STAT2 expression relative to GAPDH expression in the same samples. Shown are the means ± standard error of two independent experiments. NS: non-significant. (F) TRFs were infected like described in D and at 48 hours p.i. RNA was harvested to quantify IRF9 and ORF63 mRNA expression by qPCR. Data were normalized to the level of GAPDH mRNA expression in each sample. IRF9 expression is shown as delta cycle threshold (ΔCt). ORF63 expression is shown as relative expression compared to cells incubated with 1000 ng/ml Dox. Shown is a representative experiment of two independent experiments with three replicates/sample each.</p

SVV prevents IFN-mediated phosphorylation of STAT2 and reduces expression levels of STAT2 and IRF9.

<p>(A) TRFs were infected with SVV.eGFP (ratio 5:1) for 48 hours and stimulated for 20 minutes with 5000 U/ml uIFN. Lysates were analyzed for expression of various members (non-phosphorylated as well as phosphorylated (p)) of the JAK-STAT pathway using SDS-PAGE and western blot with antibodies specific for the indicated proteins. ORF31 expression confirmed productive viral infection and GAPDH was used as a loading control. (B) Relative expression of STAT1, STAT2 and IRF9 in SVV-infected cells compared to unstimulated mock-infected cells (set at 100%). Shown are the averages of four independent experiments. (C) Mock- and SVV.eGFP-infected cells (ratio 5:1) were stimulated with 5000 U/ml uIFN at 48 hours p.i. for 20 minutes. Cytoplasmic and nuclear fractions were isolated and analyzed for IRF9 expression by SDS-PAGE and western blotting. An antibody directed against ORF62 confirmed productive SVV-infection. Purity of fractionation was confirmed using GAPDH (cytosolic) and p84 (nuclear). Results from one of three three independent experiments is shown.</p

ORF63 induces proteasomal-degradation of IRF9, but does not affect IRF1 or IRF3.

<p>(A) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 in the presence of 1000 ng/ml Dox (-) or 1 ng/ml Dox (+) for 48 hours. During the last 16 hours of infection cells were incubated with increasing concentrations the proteosomal inhibitor MG132. Lysates were stained for IRF9 and ORF63 levels by SDS-PAGE ans western blot. The GAPDH signal was used a loading control. (B) The ratio of IRF9 to GADH expression in MG132-treated (10 μM) control and ORF63-expressing cells is presented. Shown are the means ± standard error of the average of 4 independent experiments, * indicate P-value of <0.01. (C) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 in the presence of the indicated amounts of Dox. At 48 hours p.i. cells were lysed and stained for the indicated proteins using specific antibodies. The GAPDH signal was used a loading control.</p

IFN-induced nuclear translocation of STAT is blocked in SVV-infected cells.

<p>(A) TRFs were infected with SVV.eGFP (ratio 10:1) and at 48 hours p.i., the cells were stimulated with 5000 U/ml uIFN for 40 minutes. Cells were fixed with 4% paraformaldehyde, permeablized and stained for STAT2 using a specific antibody. SVV infection (green) and STAT2 localization (red) were visualized by immunofluorescence microscopy. Insert shows enlargement of the outlined area. (B) Cytoplasmic and nuclear fractions were isolated from mock- and SVV.eGFP (ratio 5:1)-infected Vero cells that were stimulated with 5000 U/ml uIFN for 40 minutes at 48 hours p.i.. The fractions were analyzed for STAT2 expression by SDS-PAGE and western blotting. An antibody directed against ORF62 confirmed productive SVV-infection. Fraction purity was confirmed using GAPDH (cytosolic) and p84 (nuclear). Results from one of three independent experiments is shown.</p

The characteristics of a conditionally ORF63/ORF70-expressing mutant virus.

<p>(A) The SVV genome consists of unique long (UL) and a unique short (US) segments and each of them being bound by inverted repeat sequences, TRL, IRL and IRS and TRS respectively. The left end of the SVV genome contains an additional invert repeat sequence. Using a recombinant SVV BAC we generated an SVV mutant in which we fused the destabilizing dihydrofolate reductase (DHFR) to the C-terminus of both ORF63 and ORF70 (63-DHFR). (B) Mono layers of Vero cells were infected with wild type or ORF63/70-DHFR SVV in the presence of 10 μM TMP and harvested at 3, 24, 48, 72, 96, 120, 144 and 168 hours p.i. The titer of each virus was determined at each time point by plaque assay. (C) Vero cells infected with ORF63/70-DHFR SVV were cultured in the presence of 10 μM TMP (first panel), after which the virus was passaged two times on Vero cells in the absence of TMP. In the third panel, TMP was added again to the culture. Viral plaques were identified by staining with crystal violet. (D) TRFs were infected with wild type and ORF63/70-DHFR SVV for 48 hours and stimulated with 5000 U/ml uIFN for 20 minutes. Cells were lysed and expression of IRF9, STAT2 and phosphorylated STAT2 (pSTAT2) was confirmed by SDS-PAGE and western blot using specific antibodies. Lysates were specifically stained for ORF31 and ORF63 to confirm infection. GAPDH was used a loading control. (E) TRFs were infected with ORF63/70-DHFR SVV for 48 hours in the presence of the indicated concentrations of TMP. Expression of IRF9, ORF63 and ORF31 were detected SDS-PAGE and western blot using specific antibodies. GAPDH was used as a loading control. One of three independent experiments is shown.</p

ORF63 inhibits IRF9-enhanced JAK-STAT signaling in HEK 293T cells.

<p>HEK 293T cells were co-transfected with 1 μg of a plasmid encoding ISRE-luciferase, and the indicated amounts of plasmids encoding SVV ORF63 (pORF63) and rhesus IRF9 (pIRF9). At 24 hours post transfection, luciferase expression was measured. In A and C the cells were stimulated with 5000 U/ml uIFN for 6 hours before measuring luciferase activity. The relative firefly luminescence was determined by calculating the level of firefly luciferase per cell. One representative experiment of two or three independent experiments is shown.</p

IRF9 is required for efficient ISG induction and overexpression of IRF9 overcomes JAK-STAT inhbition by ORF63.

<p>(A) IRF9 expression levels were analyzed in THF-ISRE control cells and cells expressing shRNA V3LHS 322329 (shRNA1), V3LHS 322332 (shRNA2) or V2LHS 69847 (shRNA3) by SDS PAGE and western blot. (B) THF-ISRE cells were infected with AdORF63 at MOI 20 and AdTA at MOI 10 in the presence of the indicated amounts of Dox. THF-ISRE control and shRNA-expressing cells were infected with AdTA at MOI 30. At 48 hours p.i. all cells were lysed and ORF63 and IRF9 levels in the lysates were determined by SDS-PAGE and western blot using specific antibodies. GAPDH was used as a loading control. (C) RNA was isolated from THF-ISRE expressing IRF9-specific siRNAs that were stimulated for 4 or 8 hours. RT-PCR and qPCR were used to determine ISG54 expression in all cells. Data were normalized by the level of GAPDH mRNA expression in each sample and are shown as the relative fold induction. Shown are the means ± standard error of three replicates. One of two representative experiments is shown. (D) THF-ISRE cells expressing the IRF9-specific siRNAs were stimulated with 1000 U/ml uIFN for 4 or 8 hours and lysates were analyzed for ISG54 expression using SDS-PAGE and western blot.</p