Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo

Abstract

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) vhs null mutant 333d41 or the vhs rescue 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-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) 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-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.

FIG.1.

Pathogenesis of vhs-deficient HSV-2 is not restored in SCID mice. Groups of four BALB/c and congenic CB.17-SCID mice were infected i.vag. with 2 × 10⁶ PFU of vhs-deficient HSV-2, strain 333d41, or the vhs rescue virus 333d41^R. (A) Replication in the genital mucosa of BALB/c and SCID mice. The titer of virus in vaginal swab samples was determined by standard plaque assay. Data points represent the geometric mean ± standard error of the mean (SEM). (B) Severity of genital and neurological disease in BALB/c and SCID mice. Mice were scored for signs of disease as described in Materials and Methods. Values represent the arithmetic mean ± SEM. (C) Replication in the nervous systems of BALB/c and SCID mice. Mice were sacrificed 6 d postinfection. Brain, brainstem, and spinal cord tissues were disrupted by bead beating. Viral titers were determined by standard plaque assay. The dashed line indicates the limit of detection. Bars represent the geometric mean ± SEM.

FIG. 2.

Replication in the genital mucosa of 129 and IFN-α/βR^−/− mice. Groups of mice were infected i.vag. with 2 × 10⁶ PFU of the indicated HSV-2 strains. The titer of virus in vaginal swab samples of 129 mice (n = 10 to 12) (A) and IFN-α/βR^−/− mice (n = 6 to 8) (B) was determined by standard plaque assay; for ΔTK⁻, n = 5. Data points represent the geometric mean ± standard error of the mean. The dashed line indicates the limit of detection. *, P = 0.0178; **, P < 0.0001 (for 333d41 compared with 333d41^R).

FIG. 3.

Severity of genital and neurological disease in 129 and IFN-α/βR^−/− mice. Mice as described in the legend for Fig. 2 were scored for signs of disease. Values for 129 (A) and IFN-α/βR^−/− (B) mice represent the arithmetic mean ± standard error of the mean. **, P < 0.001, as determined by Kruskall-Wallis nonparametric test for 333d41 compared with 333d41^R.

FIG. 4.

Replication in the nervous systems of 129 and IFN-α/βR^−/− mice. Mice as described in the legend for Fig. 2 were sacrificed on day 6 postinfection. Brain, brainstem, and spinal cord tissues of 129 (A) and IFN-α/βR^−/− (B) mice were disrupted by bead beating. Viral titers were determined by standard plaque assay. The dashed line indicates the limit of detection. *, P = 0.032 to 0.012; **, P < 0.0001 for 333d41 compared with 333d41^R as determined by t test. n = 9 to 11 for 129 samples; n = 5 for IFN-α/βR^−/− samples due to deaths of mice. For ΔTK⁻, n = 5.

FIG. 5.

Pathogenesis of 333d41 is not restored in PKR^−/− mice. Groups of seven to nine wild-type 129 and PKR^−/− mice were infected i.vag. with 2 × 10⁶ PFU of SB5 or 333d41. n = 4 for 129/SB5. (A) The titer of virus in vaginal swab samples was determined by standard plaque assay. Data represent the geometric mean ± standard error of the mean (SEM). (B) Mice were scored for signs of disease as described in the legend for Fig. 3. Values represent the arithmetic mean ± SEM. (C) Viral titers in brain, brainstem, and spinal cord tissues were determined as described in the legend for Fig. 4. Dashed lines indicate limits of detection.

FIG. 6.

Pathogenesis of vhs-deficient HSV-1 is not restored in IFN-α/βR^−/− mice. Groups of four to five 129 and IFN-α/βR^−/− mice were infected i.vag. with 2 × 10⁶ PFU of HSV-1 KOS or vhs-deficient UL41NHB. (A) Replication in the genital mucosa. The titer of virus in vaginal swab samples was determined by standard plaque assay. Values represent the geometric mean ± the standard error of the mean (SEM). The dashed line indicates the limit of detection. (B) Severity of genital and neurological disease. Mice were scored for signs of disease as described in Materials and Methods. Values represent the arithmetic mean ± SEM.