Murine Gammaherpesvirus 68 LANA Is Essential for Virus Reactivation from Splenocytes but Not Long-Term Carriage of Viral Genome

ABSTRACT ORF73, which encodes the latency-associated nuclear antigen (LANA), is a conserved gamma-2-herpesvirus gene. The murine gammaherpesvirus 68 (MHV68) LANA (mLANA) is critical for efficient virus replication and the establishment of latent infection following intranasal inoculation. To test whether the initial host immune response limits the capacity of mLANA-null virus to traffic to and establish latency in the spleen, we infected type I interferon receptor knockout (IFN-α/βR−/−) mice via intranasal inoculation and observed the presence of viral genome-positive splenocytes at day 18 postinfection at approximately 10-fold-lower levels than in the genetically repaired marker rescue-infected mice. However, no mLANA-null virus reactivation from infected IFN-α/βR−/− splenocytes was observed. To more thoroughly define a role of mLANA in MHV68 infection, we evaluated the capacity of an mLANA-null virus to establish and maintain infection apart from restriction in the lungs of immunocompetent mice. At day 18 following intraperitoneal infection of C57BL/6 mice, the mLANA-null virus was able to establish a chronic infection in the spleen albeit at a 5-fold-reduced level. However, as in IFN-α/βR−/− mice, little or no virus reactivation could be detected from mLANA-null virus-infected splenocytes upon explant. An examination of peritoneal exudate cells (PECs) following intraperitoneal inoculation revealed nearly equivalent frequencies of PECs harboring the mLANA-null virus relative to the marker rescue virus. Furthermore, although significantly compromised, mLANA-null virus reactivation from PECs was detected upon explant. Notably, at later times postinfection, the frequency of mLANA-null genome-positive splenocytes was indistinguishable from that of marker rescue virus-infected animals. Analyses of viral genome-positive splenocytes revealed the absence of viral episomes in mLANA-null infected mice, suggesting that the viral genome is integrated or maintained in a linear state. Thus, these data provide the first evidence that a LANA homolog is directly involved in the formation and/or maintenance of an extrachromosomal viral episome in vivo, which is likely required for the reactivation of MHV68.

The Murine Gammaherpesvirus 68 M2 Gene Is Required for Efficient Reactivation from Latently Infected B Cells

ABSTRACT Murine gammaherpesvirus 68 (γHV68) infection of mice provides a tractable small-animal model system for assessing the requirements for the establishment and maintenance of gammaherpesvirus latency within the lymphoid compartment. The M2 gene product of γHV68 is a latency-associated antigen with no discernible homology to any known proteins. Here we focus on the requirement for the M2 gene in splenic B-cell latency. Our analyses showed the following. (i) Low-dose (100 PFU) inoculation administered via the intranasal route resulted in a failure to establish splenic B-cell latency at day 16 postinfection. (ii) Increasing the inoculation dose to 4 × 105 PFU administered via the intranasal route partially restored the establishment of B-cell latency at day 16, but no virus reactivation was detected upon explant into tissue cultures. (iii) Although previous data failed to detect a phenotype of the M2 mutant upon high-dose intraperitoneal inoculation, decreasing the inoculation dose to 100 PFU administered intraperitoneally revealed a splenic B-cell latency phenotype at day 16 that was very similar to the phenotype observed upon high-dose intranasal inoculation. (iv) After low-dose intraperitoneal inoculation, fractionated B-cell populations showed that the M2 mutant virus was able to establish latency in surface immunoglobulin D-negative (sIgD−) B cells; by 6 months postinfection, equivalent frequencies of M2 mutant and marker rescue viral genome-positive sIgD− B cells were detected. (v) Like the marker rescue virus, the M2 mutant virus also established latency in splenic naive B cells upon low-dose intraperitoneal inoculation, but there was a significant lag in the decay of this latently infected reservoir compared to that seen with the marker rescue virus. (vi) After low-dose intranasal inoculation, by day 42 postinfection, latency was observed in the spleen, although at a frequency significantly lower than that in the marker rescue virus-infected mice; by 3 months postinfection, nearly equivalent levels of viral genome-positive cells were observed in the spleens of marker rescue virus- and M2 mutant virus-infected mice, and these cells were exclusively sIgD− B cells. Taken together, these data convincingly demonstrate a role for the M2 gene product in reactivation from splenic B cells and also suggest that disruption of the M2 gene leads to dose- and route-specific defects in the efficient establishment of splenic B-cell latency.

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 333d41R. 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-2 vhs mutants 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, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41R. 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-α/βR−/− 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-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 the vhs mutation, 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-1 vhs null 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.