HSV-2 inhibits type-I interferon signaling via multiple complementary and compensatory STAT2-associated mechanisms - PubMed (original) (raw)

HSV-2 inhibits type-I interferon signaling via multiple complementary and compensatory STAT2-associated mechanisms

Ravi-Kumar Kadeppagari et al. Virus Res. 2012 Aug.

Abstract

Type-I interferon (IFN)-mediated responses are a crucial first line of defense against viral infections and are critical for generating both innate and adaptive immunity. Therefore, viruses have necessarily evolved mechanisms to impede the IFN response. HSV-2 was found to completely abolish type-1 IFN-mediated signaling via multiple STAT2-associated mechanisms. Although the extent and kinetics of this inactivation were indistinguishable between the various cell-lines examined, there were distinct differences in the mechanisms HSV-2 employed to subvert IFN-signaling among the cell-lines. These mechanistic differences could be segregated into two categories dependent on the phase of the HSV replicative cycle that was responsible for this inhibition: (1) early phase-inhibited cells which exhibited abrogation of IFN-signaling prior to viral DNA replication; (2) late phase-inhibited cells where early phase inhibition mechanisms were not functional, but viral functions expressed following DNA replication compensated for their ineffectiveness. In early phase-inhibited cells, HSV-2 infection targeted STAT2 protein for proteosomal degradation and prevented de novo expression of STAT2 by degrading its mRNA. In contrast, HSV-2 infected late phase-inhibited cells exhibited no apparent changes in STAT2 transcript or protein levels. However, in these cells STAT2 was not activated by phosphorylation and failed to translocate to the cell nucleus, thereby preventing transactivation of antiviral genes. In primary human fibroblasts, HSV-2 failed to fully degrade STAT2 and therefore, both early and late phase mechanisms functioned cooperatively to subvert IFN-mediated antiviral gene expression. Taken together, these results indicate the importance that HSV-2 has assigned to STAT2, investing significant genomic currency throughout its replicative lifecycle for continuous targeted destruction and inhibition of this protein.

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Figures

Figure 1

HSV-2 infection abrogates IFN-mediated induction of ISG expression in primary HDFa cells. Western blot analysis of ISG expression in HDFa cells that were mock-infected or infected with HSV-2 and subsequently mock-treated or treated with IFNβ. Blots were probed with antibodies to STAT1, Mx1, ISG15, HSV1/2 gB, or actin.

Figure 2

HSV-2 specifies mechanisms that inhibit type-I IFN signaling both early and late in the replicative cycle. (A & B) ISRE promoter-reporter analysis of HSV-2 mediated inhibition of type-I IFN signaling in mock- (blue bars) or HSV-2- (red bars) infected 293A and HeLa (early phase-inhibited) cells (A) or 293α/β and C33A (late phase-inhibited) cells (B). Treatment of various HSV-2 infected cell-lines with the viral DNA replication inhibitors PAA or acyclovir segregates the replicative phase that HSV-2 utilizes to mediate abrogation of IFN signaling. (C & D) Kinetics of HSV-2 inhibition of IFN-mediated activation of the ISRE promoter in early phase-inhibited (C) and late phase-inhibited (D) cells. Red arrow indicates the time of HSV-2 infection (0h) relative to addition of IFNβ at −8, 0, 4, 8, 12, and 16h.

Figure 3

Semi-quantitative RT-PCR of cellular transcripts for the members of the IFN-associated ISGF3 complex (STAT2, STAT1 and IRF9) in mock- or HSV-2-infected early phase-inhibited (A) and late phase-inhibited (B) cells at 0, 4, 8, and 16 hpi.

Figure 4

Cell-line dependent analysis of the influence HSV-2 infection has on the STAT2 3′UTR. (A) Diagram of the 3′UTR indicator vector constructs that specify the 3′UTR of either STAT1 or STAT2 cloned downstream of a luciferase reporter open reading frame. Luciferase assays were performed following transfection of either 0.1ug or 1.0 ug into the indicated cell lines. HSV-2-mediated inhibition through the 3′UTR is measured by normalized luciferase activity in infected cells relative to the activity of the construct in uninfected cells. The control parental luciferase vector with no 3′UTR was set to an arbitrary value of 1 and the fold inhibition of each of the 3′UTR constructs was determined relative to this value. (B) In early phase-inhibited 293A cells, HSV-2 infection inhibits luciferase activity of transcripts that specify the STAT2 3′UTR (black bars), but not the STAT1 3′UTR (gray bars). (C) In late phase-inhibited C33A cells, HSV-2 infection does not affect the relative luciferase activity from transcripts specifying either the STAT1 or STAT2 3′UTR. (D & E) The relative effect of HSV-2 infection on luciferase expression was determined for transcripts that specified no 3′ UTR (Luciferase) or either the STAT1 3′UTR or STAT2 3′UTR in early phase-inhibited 293A or late phase-inhibited C33A cells.

Figure 5

HSV-2 infection specifically affects STAT2 protein levels in early phase-inhibited (A) but not late phase-inhibited (C) 293α/β cells. HSV-2 infected 293A cells were analyzed by western blot for levels of STAT1, STAT2, IRF9, HSV gB and actin expression at 0, 4, 8, and 16 hpi. (B) In early phase-inhibited 293A cells, abrogation of HSV-2 DNA replication by PAA does not affect HSV-2-mediated loss of STAT2 expression. The same results were observed with acyclovir treatment (data not shown). (C) In late phase-inhibited 293α/β cells, STAT2 protein levels are unaffected by HSV-2 infection.

Figure 6

Cellular STAT2 has an exceedingly stable half-life and is therefore targeted for proteosomal degradation in HSV-2 infected cells. (A) Assessment of the stability of cellular STAT2 in 293A cells following Actinomycin D (ActD) and Cyclohexamide (CHX) inhibition of de novo STAT2 protein expression. (B) Western analysis of HSV-2’s ability to alter cellular STAT2 protein levels in the presence (+) or absence (−) of proteosome inhibitors.

Figure 7

In HSV-2 infected late phase-inhibited cells, which do not degrade cellular STAT2 protein, HSV-2 infection inhibits STAT2 phosphorylation. C33A (A) or 293α/β (B) cells were mock- or HSV-2-infected and at 16 hpi were mock-treated (Mock Tx) or treated with IFNβ or IFNγ and subsequently analyzed by western for phosphorylation of STAT1 and STAT2. Parallel experiments were conducted in the presence of the viral DNA replication inhibitor PAA. Identical results were observed with acyclovir treatment (data not shown). (C) The kinetics of HSV-2 inhibition of IFNβ-mediated STAT2 phosphorylation was examined by treating HSV-2 infected cells at 0, 4, 8, and 16 hpi.

Figure 8

HSV-2 late phase inhibition of STAT2 phosphorylation abrogates translocation of STAT2 from the cytoplasm to the nucleus. (A) Mock-infected or HSV-2-infected 293α/β cells were mock-treated or treated with IFNβ, separated into cytoplasmic and nuclear fractions, and analyzed by western for STAT2 (αSTAT2) and phosphorylated STAT2 (αPO4 STAT2) proteins. Parallel experiments were conducted in the presence of the viral DNA replication inhibitors PAA or acyclovir. (B) Immunofluorescence analysis of STAT2 (green) and phosphorylated STAT2 (green) 293α/β cellular localization under the indicated conditions. Nuclei are demarcated in blue by Hoescht staining.

Figure 9

In primary HDFa cells, HSV-1 and HSV-2 infection induces only partial but specific loss of STAT2 expression, but compensates by inhibiting STAT2 phosphorylation. (A) Western blot analysis of HDFa cells either mock infected or infected with HSV-2 or HSV-1. To determine relative levels of protein expression, blots were probed with antibodies to STAT2, STAT1, IRF9, HSV1/2 gB, or actin. (B) The ability of IFNβ to induce STAT1 or STAT2 phosphorylation in mock infected or in the context of an HSV-1 or HSV-2 infection was determined by western blot analysis.

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HSV-2 inhibits type-I interferon signaling via multiple complementary and compensatory STAT2-associated mechanisms - PubMed (original) (raw)