This study is focused on the KSHV which has been found in KS and in biopsies of some non-Hodgkin's lymphoma. The BCBL line, which is latently infected with both KSHV and EBV, is the only known cell culture source for KSHV grown in cell culture, as well as the only human cells known to be latently infected with two human herpes viruses. Preliminary studies demonstrating that the BCBL line can be induced to express KSHV lytic cycle peptides and amplify KSHV DNA are the basis of this application.
The specific aims are: 1. To develop serologic markers to identify lytic cycle proteins; 2. To isolate infectious KSHV; and 3. To define the latent to lytic cycle switch of KSHV in BCBL cells. The approach to develop serologic markers will be to do immunoblotting and immunofluorescence assays using BCBL cells and patient-derived antibody- containing sera. Upon recognition of the lytic cycle-associated proteins, cloning and sequence analyses of the proteins will be done. The approach towards isolating infectious virus will initially focus on the BCBL line, then proceed to patient source material. Target cells already tested included B and T cell lines, which all proved negative. For this proposed work, the MRC5 cell strain, primary cultures of dermal fibroblasts, and umbilical vein endothelial cells will be tested, as will some lines of monkey kidney cells. Work with the BCBL line will include experiments to determine the size and complexity of the KSHV genome carried by the cell line. It will also capitalize on the observation the KSHV can co-infect B cells with EBV, by using EBV immortalized cells as one of the target systems for virus isolation. The approach to understanding the latent to lytic cycle switch will be to identify inducers, then quantitate numbers of cells in populations that are induced and as well as the quantitative and qualitative production of KSHV polypeptides and mRNAs. Included in this approach is the use of representational difference analysis and subtraction cDNA libraries. This will lead to the derivation of genomic and cDNA clones of KSHV which would be used to define immediate early and early KSHV gene expression in the BCBL line. These tools, combined with existing tools available for the study of EBV, will help determine if KSHV and EBV are under coordinate or distinct control.
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