Primary infection with varicella-zoster virus (VZV) causes chickenpox, and reactivation of the virus from latency results in zoster. The purpose of this project is to study the molecular pathogenesis and latency of VZV, to determine the mechanism for attenuation of the current VZV vaccine, and to identify receptors that the virus uses to infect cells. ? ? The major VZV gene expressed during latency is gene 63. Gene 63 RNA and protein have been detected in latently infected human and animal ganglia. Cells infected with a gene 63 deletion mutant yielded very low titers of cell-free virus and produced few fully processed virions compared with those infected with wild-type virus. Microarray analysis of cells infected with the deletion mutant showed a 4-fold increase expression of the major viral transactivator (gene 62) which turns on multiple other viral genes, compared with cells infected with wild-type virus. Similarly a 3-fold increase in gene 62 protein was detected with cells infected with the ORF63 mutant virus. Cells infected with gene 63 mutants that were impaired for latency showed increased expression of VZV gene 62, while cells infected with gene 63 mutants that were not impaired for latency expressed gene 62 at levels similar to those infected with wild-type virus. Thus, the ability of gene 63 to down-regulate expression of gene 62, the major viral transactivator, may play an important role in virus latency and limiting viral gene expression.? ? We studied differences between the VZV Oka vaccine strain (V-Oka), currently licensed for prevention of varicella and zoster, and its parent virus from which it was derived (P-Oka). Prior studies indicated that gene 62 had the greatest number of nucleotide and amino acid differences when the two viruses were compared. Comparison of VZV gene expression between V-Oka and P-Oka showed that cells infected with V-Oka expressed lower levels of RNAs for genes 62, 65, 66, and 67, and higher levels of gene 41 RNA, compared to cells infected with P-Oka. The level of genes 62, 65, and 66 protein was confirmed to be lower in cells infected with V-Oka compared to P-Oka. These results indicate that while the sequence of ORF62 has a large number of amino acid differences in the two viruses, these changes alone are unlikely to account for attenuation of V-Oka. ? ? We have identified a cellular protein that interacts with a VZV glycoprotein, gE. We produced a recombinant soluble form of gE and found that it bound to a cellular protein in human melanoma cells. The cellular protein was identified as insulin-degrading enzyme (IDE). While IDE is predominantly present inside the cells, we and others have shown that a portion of IDE is present on the surface of cells. We further confirmed that IDE interacts with VZV in virus-infected cells.? ? We used a number of assays in ?loss of function? tests to show that IDE is important for entry of VZV into cells. Treatment of cells with antibody to IDE, or with soluble IDE extracted from human liver reduced infectivity of cell-free VZV, and reduced cell-to-cell spread of the virus. Treatment of cells with a molecule that blocks expression of IDE (small interfering RNA) inhibited infection with cell-free virus and reduced cell-to-cell spread of virus. Finally bacitracin, an antibitoic that inhibits IDE activity, blocked the ability of gE to bind to IDE, and reduced infection with cell-free virus and cell-to-cell spread of virus.? ? We also performed a number of ?gain of function? tests to determine if additional human IDE could enhance infection in nonhuman cells that do not permit VZV infection. Expression of human IDE in mouse or hamster cells enhanced infectivity of VZV. Expression of human IDE in hamster cells also enhanced the binding of radiolabeled cell-free VZV to the cells. Taken together these results indicate that IDE serves as a cellular receptor for both cell-free and cell-associated VZV.
Showing the most recent 10 out of 21 publications
