Varicella-zoster virus (VZV) causes chickenpox and shingles. Virus infection of cells is known to trigger several signaling pathways that are important for cell proliferation and prevention of programmed cell death (also known as apoptosis). We have used a virus genome wide approach to identify functions of VZV proteins. Nearly all of the 71 VZV genes were individually expressed in tissue culture cells along with a reporter system that responds to specific signaling proteins. Using this approach, last year we showed that VZV ORF12 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (a cell signaling pathway) to regulate cell cycle progression (important for cell division). Since VZV replicates in both dividing (keratinocytes) and non-dividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body. This year we found that VZV triggers the phosphorylation of two proteins, Bim and BAD, that are important for inducing programmed cell death (also known as apoptosis). Phosphorylation of these proteins reduces their ability to cause cell death. VZV-infected cells that were knocked-down for expression of Bim survived longer and produced higher titers of virus compared with wild-type cells infected with the virus. In contrast, VZV-infected cells that over-expressed Bim showed reduced survival and reduced virus replication. Inhibition of caspase activity in cells overexpressing Bim restored levels of virus replication to those seen in wild-type cells. Mammalian cells activate DNA damage response pathways in response to virus infections. H2AX and ATM are activated and recruited to the site of double-stranded DNA breaks and are important for repairing the DNA. We found that VZV-infected cells had elevated levels of phosphorylated H2AX and ATM compared with uninfected cells. Cells infected with VZV deleted for ORF61 or ORF63, but not ORF67, had higher levels of phosphorylated H2AX and ATM compared with cells infected with wild-type virus. We have also been developing a more sensitive assay to detect antibody to VZV in recipients of the varicella vaccine. We are comparing our assay using sera from persons who received the vaccine, with results obtained from the fluorescent antibody to membrane antigen (FAMA) assay, to determine the sensitivity and specificity of our assay.
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