Anti-CD8 impairs clearance of herpes simplex virus from the nervous system: implications for the fate of virally infected neurons.

The role of CD8+ T cells in resistance to herpes simplex virus (HSV) was examined. After cutaneous inoculation, HSV spreads to the peripheral nervous system (PNS) where it replicates in ganglionic neurons. In normal mice, replication of virus in the PNS was rapidly terminated and evidence of neuronal destruction, assessed by a quantitative histological assay, was sparse. Clearance of infectious virus was impaired, and a strikingly high proportion of ganglionic neurons was killed, in mice treated with an antibody that depleted them of CD8+ T cells. These results suggest that CD8+ T cells play an important role in maintaining the integrity of the sensory nervous system during primary infection with HSV. Therefore, viral epitopes recognized by CD8+ T cells and restricting class I major histocompatibility complex genes are, in principle, implicated as interacting genetic determinants of neurovirulence.

Upregulation of class I major histocompatibility complex gene expression in primary sensory neurons, satellite cells, and Schwann cells of mice in response to acute but not latent herpes simplex virus infection in vivo.

Major histocompatibility complex (MHC) deficiency is typical of almost all resident cells in normal neural tissue. However, CD8+ T cells, which recognize antigenic peptides in the context of class I MHC molecules, are known to mediate clearance of herpes simplex virus (HSV) from spinal ganglia of experimentally infected mice, leading to the hypothesis that class I expression in the peripheral nervous system must be upregulated in response to HSV infection. In addressing this hypothesis it is shown, in BALB/c (H-2d) mice, that normally deficient class I transcripts transiently accumulate in peripheral nerve Schwann cells, ganglionic satellite cells, and primary sensory neurons, indicating that in each of these cell types class I expression is regulated at the transcriptional level in vivo. Furthermore, for 3-4 wk after infection, H-2Kd/Dd antigens are expressed by satellite and Schwann cells but not neurons, suggesting additional posttranscriptional regulation of class I synthesis in neurons. Alternatively, the class I RNAs induced in neurons may not be derived from classical class I genes. Factors regulating H-2 class I expression emanate from within infected ganglia, probably from infected neurons themselves. However, induction of class I molecules was not maintained during latency, when viral gene expression in neurons is restricted to a single region within the virus repeats. These data have implications for the long-term survival of cells in HSV-infected neural tissue.

Use of mRNA differential display to study the action of lymphocyte subsets in vivo and application to a murine model of herpes simplex virus infection.

Depletion of lymphocyte subsets in vivo using monoclonal antibodies against cell surface markers has helped to define the roles for these subsets in many immune processes. However, in some cases the mechanisms through which these lymphocytes act remain partially elucidated or completely unknown. A new approach to these biological problems is the use of transcriptional analyses to find mRNAs whose abundance in tissues is altered by depletion of lymphocyte subsets. We have verified the use mRNA differential display (DD) for this purpose and applied it in a study of CD8(+) lymphocyte mediated clearance of herpes simplex virus (HSV) from the nervous systems of experimentally infected mice. The results of the differential displays and characterisation of a large mRNA identified using this strategy are presented.

Anti-CD8 treatment alters interleukin-4 but not interferon-gamma mRNA levels in murine sensory ganglia during herpes simplex virus infection. Brief report.

Clearance of herpes simplex virus (HSV) from sensory ganglia of infected mice is dependent on CD8(+) cells but not on the death of infected neurons. The mechanism of action of CD8(+) cells in HSV infected ganglia is therefore unknown. The determine which cytokines might be involved in the CD8(+) cell dependent response to ganglionic HSV infection, IL-2, IL-4, IL-6, IL-10, and IFN-gamma mRNA levels were measured in infected ganglia from immunocompetent and anti-CD8 treated mice. Anti-CD8 treatment increased the abundance of mRNA encoding IL-4, and, to a lesser extent, IL-2, and IL-6. Significantly, IFN-gamma mRNA was not affected.

The vaccinia virus superoxide dismutase-like protein (A45R) is a virion component that is nonessential for virus replication.

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.

Vaccinia virus gene B7R encodes an 18-kDa protein that is resident in the endoplasmic reticulum and affects virus virulence.

This paper presents a characterisation of vaccinia virus (VV) gene B7R that was predicted to encode a polypeptide of 182 amino acids with an N-terminal signal peptide. In vitro transcription and translation analysis showed the B7R gene product was a 21-kDa protein that, in the presence of microsomes, was processed into an 18-kDa mature form. The 18-kDa form associated with the microsomal membranes and was within the lumen of the vesicle where it was inaccessible to exogenous protease or an antibody raised against the B7R C terminus. Within VV-infected cells, the 18-kDa form of B7R was detected late during infection in the endoplasmic reticulum where it colocalised with protein disulphide isomerase. The B7R protein was detected neither in the culture supernatant nor associated with virus particles. A virus deletion mutant lacking the B7R gene and a revertant virus were constructed. Compared to wild-type and revertant viruses, the deletion mutant replicated normally in cell culture and had unaltered virulence in a murine intranasal model of infection. However, the deletion mutant was attenuated in a murine intradermal model where it induced a smaller lesion than the control viruses.