The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2)vhsnull mutant 333d41 or thevhsrescue virus 333d41^R. vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-α/β). The attenuation of HSV-2vhsmutants could reflect their failure to suppress IFN-α/β-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-α/β receptor-deficient (IFN-α/βR^−/−) mice were infected i.vag. with wild-type virus,vhsnull mutants 333-vhsB or 333d41, or thevhsrescue virus 333d41^R. Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type orvhsrescue viruses, they were restored to nearly wild-type levels of replication in IFN-α/βR^−/−mice over the first 2 days postinfection. Only wild-type andvhsrescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-α/βR^−/−mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-α/βR^−/−mice. Restoration was specific for thevhsmutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-α/βR^−/−mice. Furthermore, the defect in the IFN-α/β response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-α/β-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-α/β response in vivo, because an HSV-1vhsnull mutant did not recover replication and virulence after i.vag. inoculation into IFN-α/βR^−/−mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-α/β-mediated antiviral response.

Herpes simplex virus mutants lacking the vhs protein are severely attenuated in animal models of pathogenesis and exhibit reduced growth in primary cell culture. As a result of these properties, viruses withvhsdeleted have been proposed as live-attenuated vaccines. Despite these findings and their implications for vaccines, the mechanisms by which vhs promotes infection in cell culture and in vivo are not understood. In this study we demonstrate that vhs-deficient viruses replicate to reduced levels in interferon (IFN)-primed cells and that this deficit has both IFN-dependent and IFN-independent components. Furthermore, vhs-defective viruses induce increased and physiologically active levels of IFN, increased amounts of IFN-stimulated transcripts, and more phosphorylated eIF2α. In addition, we demonstrate greater accumulation of viral RNAs following infection with a vhs-deficient virus. This leads to the hypothesis that attenuation of viruses lacking vhs may be attributed to increased levels of double-stranded RNA, a potent pathogen-associated molecular pattern. Together these data show that vhs likely functions to reduce innate immune responses and thereby acts as a critical determinant of viral pathogenesis.

Herpes simplex type virus 2 (HSV-2) is a sexually transmitted pathogen that causes genital lesions and spreads to the nervous system to establish acute and latent infections. Systemic but not mucosal cellular and humoral immune responses are elicited by immunization of mice with a replication-defective mutant of HSV-2, yet the mice are protected against disease caused by subsequent challenge of the genital mucosa with virulent HSV-2. In this study, we investigated the role of immune serum antibody generated by immunization with a replication-defective HSV-2 vaccine prototype strain in protection of the genital mucosa and the nervous system from HSV-2 infection. Passive transfer of replication-defective virus-immune serum at physiologic concentrations to SCID or B-cell-deficient mice had no effect on replication of challenge virus in the genital mucosa but did significantly reduce the incidence and severity of genital and neurologic disease. In contrast, B-cell-deficient mice immunized with replication-defective HSV-2 were able to control replication of challenge virus in the genital mucosa, but not until 3 days postchallenge, and were not completely protected against genital and neurologic disease. Passive transfer of physiologic amounts of immune serum to immunized, B-cell-deficient mice completely restored their capacity to limit replication of challenge virus in the genital mucosa and prevented signs of genital and systemic disease. In addition, the numbers of viral genomes in the lumbosacral dorsal root ganglia of immunized, B-cell-deficient mice were dramatically reduced by transfer of immune serum prior to challenge. These results suggest that there is an apparent synergism between immune serum antibody and immune T cells in achieving protection and that serum antibody induced by vaccination with replication-defective virus aids in reducing establishment of latent infection after genital infection with HSV-2.

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8⁻virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs⁻/ICP8⁻HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs⁻/ICP8⁻double mutant or vhs⁻or ICP8⁻single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs⁻/ICP8⁻mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs⁻virus and exceed those of mice immunized with the ICP8⁻single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs⁻/ICP8⁻and vhs⁻virus-vaccinated mice. Moreover, the double deleted vhs⁻/ICP8⁻virus protected mice better in all respects than the single deleted ICP8⁻mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.