Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death.

Host inflammatory mediators, such as interferons, play a protective role in infection, but the mechanism is undefined and may differ between tissue compartments. To determine whether interferon-gamma (IFN-gamma) elicitation prevents destructive encephalitis in herpes simplex virus type 1 (HSV-1) infection of the central nervous system, IFN-gamma-knockout (GKO) mice were challenged intravitreally with HSV-1 strain F, inciting infection of the eyes and the brain. Indeed, the GKO mice showed encephalitis with ataxia, whereas nontransgenic controls remained asymptomatic. Morphology and digoxigenin labeling of DNA fragments revealed increased apoptosis in the brains of GKO mice compared with controls, although viral replication was not influenced at early stages of infection. Greater numbers of apoptotic cells in the brains of GKO mice correlated with neurological symptoms, as well as lower expression of the protective protooncogene bcl-2. Thus, IFN-gamma inhibits apoptosis, affording neuronal protection from destructive encephalitis during viral infection of the central nervous system.

Entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways.

OBLV60 is an acid-dependent syncytium-forming variant isolated from OBL21 cells persistently infected with the pH-independent mouse hepatitis virus (MHV)-4 strain. The fusion activity of OBLV60 can be strictly regulated by controlling pH and thus provides the means to definitively examine the entry of MHV into cells by endosomal and nonendosomal pathways. Shortly after high multiplicity infection, both MHV-4 and OBLV60 were detected by electron microscopy in endosomal vesicles and were recovered from lysates of cells treated with proteinase K to remove extracellular virus. For OBLV60, but not MHV-4, exposure to lysosomotropic compounds early in infection prevented viral penetration and significantly reduced viral yields. These results suggested that both MHV-4 and OBLV60 utilized the endosomal route of entry into cells, but that MHV-4 did not require acidification of endosomal vesicles. Studies on the entry of virus through fusion at the cell surface were performed by briefly exposing surface-bound OBLV60 to a fusion-permissive pH under conditions that prevent endocytic entry. Acid treatment of surface-bound OBLV60 caused a significant increase in the yields of virus produced in cultures of fusion-sensitive Sac- or DBT cells, demonstrating entry of virus by fusion at the cell surface. No measurable increase in virus production was detected with acid treatment of OBLV60 bound to OBL21 cells, suggesting that entry at the cell surface does not occur in these cells, which are resistant to MHV-induced syncytia formation. These results raise interesting questions concerning how mechanisms of MHV entry influence the selection of fusion variants.

Spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection.

The mouse hepatitis virus (MHV) spike glycoprotein mediates attachment of the virus to the MHV receptor, the murine biliary glycoprotein (BGP) carcinoembryonic antigen. Monoclonal antibody CC1 directed against BGP specifically inhibited infection of DBT, Sac-, GT1-7, and OBL21 cells by wild-type MHV-4 and the neuron-adapted variant OBLV60. Binding to this receptor was necessary to establish infection by cell-free MHV; however, the presence of BGP was not required for infection by cell-associated virus. Cell-associated infectious induced syncytium formation on Vero and BHK cells, which lack murine BGP; this activity was not inhibited by monoclonal antibody CC1. Antibody CC1 also did not prevent syncytium formation on DBT cells, which bear BGP. In infectious center assays, the MHV-4 variant OBLV60, which exhibits acid-dependent fusion, spread to cells lacking BGP only when exposed to acidic media. Therefore, spike-mediated fusion was required for BGP-independent spread of MHV infection. Furthermore, BGP-independent, cell-associated spread of MHV-4 was prevented by monoclonal antibodies 5A13.5 and 5B19.2 directed against the spike glycoprotein, but not by other neutralizing and nonneutralizing anti-spike antibodies. Expression of spike glycoprotein by recombinant vaccinia virus resulted in fusion of BGP-negative cells; monoclonal antibodies 5A13.5 and 5B19.2 strongly inhibited spike-mediated fusion in this assay.

Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 gamma 34.5 mutants in a mouse eye model.

The herpes simplex virus type 1 (HSV-1) gamma 34.5 gene is located within a region that is transcriptionally active during latent HSV-1 infection. To determine whether the gamma 34.5 gene deletion affects latency-associated transcript (LAT) gene expression or latent HSV-1 infection, a gamma 34.5 gene deletion mutant, 1716, and a stop codon insertion mutant, 1771, were studied in the mouse eye model. Although the gamma 34.5 gene is not essential, 1716 and 1771 replicated poorly in mouse eyes and trigeminal ganglia (TG). When mice were inoculated with 1716, infectious virus was detected in eyes only on the first day post-infection (p.i.), and was not detected at any time point in TG. Following inoculation with 1771, a small amount of virus was detected in the eyes on days 2 and 4 p.i., and in the TG of one animal on day 2 p.i. Reactivation of virus from mice latently infected with 1716 (0/30 TG) and 1771 (1/20 TG) was extremely low compared with the parental strain, 17+, and appropriate rescuants (80 to 100% reactivation), even though latent 1716 DNA was detected by PCR in 50% of TG. These results differ from those obtained following footpad inoculation; in the footpad there was limited 1716 replication and reactivatable latent infection was established in some dorsal root ganglia. The data support the hypothesis that the role of gamma 34.5 may be tissue and/or cell type specific. The synthesis, processing, and stability of the 2.0 kb LAT during 1716 and 1771 replication was not affected by these mutations in the gamma 34.5 gene. However, during latent infection of 1716 in mice the LATs were not detectable in TG by Northern blot, and were present in reduced amounts (approximately 10-fold less) during 1771 latency. The LATs from 1716 were barely detectable in a few neurons by in situ hybridization. Therefore, the gamma 34.5 gene might (i) affect replication in the eye, and reduce the amount of virus available to establish latent infection, be directly involved in (ii) establishment of latency, and/or (iii) the reactivation process.

MHVR-independent cell-cell spread of mouse hepatitis virus infection requires neutral pH fusion.

Receptor-specificity is a key determinant of viral tropism. In this report, however, we have demonstrated that cell-associated spread of MHV can bypass the requirement for binding to primary receptors and thereby spread to cells that are resistant to MHV infection. Anti-receptor antibody CC1, which blocks infection by MHV virions, failed to prevent cell-associated spread of MHV to receptor-negative BHK cells or receptor-positive DBT cells. Cell-associated MHV may be utilizing an alternative, low-affinity receptor that is inadequate for functional interaction with MHV virions. Theoretically, dissemination of MHV infection through a receptor-independent, cell-associated mechanism in vivo provides the potential for broader host and tissue range, and for spread of infection despite the presence neutralizing antibodies. Receptor-independent, cell-associated spread of MHV requires neutral pH fusion capability. The low pH-dependent MHV variant OBLV60, which utilizes an endocytic route of entry, does not spread through a receptor-independent mechanism. Additionally, antiviral antibodies that block MHV spike-mediated fusion inhibited cell-associated spread of infection.

The UL55 and UL56 genes of herpes simplex virus type 1 are not required for viral replication, intraperitoneal virulence, or establishment of latency in mice.

A critical determinant for intraperitoneal virulence of Herpes Simplex Virus Type 1 (HSV-1) strain F has been ascribed to the BamHI-B fragment of the genome. This region contains two nonessential genes, UL55 and UL56, of unknown function. To investigate the contribution of these genes to viral pathogenesis, we have generated two recombinant HSV-1 viruses, inUL55 and inUL56, in which the corresponding gene was inactivated in strain F by insertion of an ICP6::lacZ cassette. Strains inUL55 and inUL56, which did not express the UL55 and UL56 RNA, respectively, were virulent in mice inoculated intraperitoneally. Strains inUL55 and inUL56 also established latent infections in the trigeminal ganglia of mice inoculated via the cornea. These results demonstrate that the HSV-1 UL55 and UL56 genes are not critical for intraperitoneal virulence or establishment of latent infection.