Herpes simplex virus 2 virion host shutoff protein interferes with type I interferon production and responsiveness

The herpes simplex virus 2 (HSV-2) virion host shutoff (vhs) protein is a ribonuclease contained in the virion tegument. vhs-deficient mutants of HSV-2 are profoundly attenuated in vivo, and we have previously shown that replication and virulence of vhs-deficient HSV-2 are largely restored to levels of wild-type virus in mice lacking the interferon alpha/beta receptor (IFNαβR-/-). This result demonstrated that HSV-2 vhs interferes with the type I IFN response, but whether vhs inhibits production of type I IFN or synthesis or function of key mediators of the IFN-induced antiviral state was not clear. Here we address these questions using primary murine embryonic fibroblasts (MEFs), which produce and respond to IFNαβ. The vhs-deficient HSV-2 strain 333d41 replicated similarly to wild-type virus (333 clone SB5) and vhs rescue virus (333d41R) after infection of MEFs at high moi, but at low moi, 333d41 replication was severely attenuated, recapitulating the attenuated phenotype of vhs-deficient HSV-2 in vivo. Replication of 333d41 at low moi was restored to levels of wild-type virus in MEFs lacking the IFNαβ receptor or when IFNαβ was neutralized, thus establishing the IFNαβ response as the sole mechanism attenuating vhs-deficient HSV-2 replication in MEFs. MEFs infected with 333d41 produced >50-fold more IFNαβ than cells infected with 333 and 333d41R. Pretreatment of MEFs with type I IFN inhibited replication of 333d41 more than 333 and 333d41R, indicating that vhs also interferes with activation of the IFNαβ-induced antiviral response. We therefore examined vhs interference with PKR and RNase L, two key mediators of the IFNαβ response. 333d41 replication was restored to wild-type levels after low moi infection of PKR-/- and RNase L-/- MEFs, and was not inhibited in PKR-/- MEFs pretreated with IFNα. Together, these observations indicate that HSV-2 vhs is a broad and potent countermeasure to the IFN-mediated antiviral response in IFN-naieve and -sensitized MEFs