Establishment of a Quiescent Infection of HSV-1 in L929 Fibroblasts using a Mitotic Inhibitor and IFN-γ
Gerald M. Alter (Committee Member), Nancy J. Bigley (Advisor), Barbara E. Hull (Committee Member)
Master of Science (MS)
The goal for this study was to determine if a quiescent infection of HSV-1 could be induced in murine fibroblasts L929 by treating them with the anti-mitotic agent 5-fluoro 2'deoxy uridine (FUDR) alone and with interferon-γ. Since neurons are post-mitotic and exhibit a lower metabolic rate than other cells, fibroblasts were treated with FUDR to induce a post-mitotic state. The cell cycle arrest of fibroblasts would decrease the thymidylate metabolism and impair HSV-1 replication. An evaluation of cytopathic effects of FUDR was used to determine the optimal concentration which arrests cell growth and inhibits viral replication. Image J program developed by NIH was used to analyze images of cultured L929 cells. In initial experiments cells showed protection from cytopathic effects of HSV-1 when treated with FUDR and IFN-γ. To determine whether the virus was in a quiescent state in L929 cells attempts were made to rescue viable virus from these cells. The FUDR+ IFN-γ + HSV-1 treated L929 cells were co-cultured with Vero cells or lysate from L929 cells was added to Vero cells. Viral plaques indicating viral rescue were observed after 48hrs.of incubation by staining the cells with crystal violet, indicating that HSV-1 was in a silent state in these treated L929 cells.
Hoechst staining was performed to detect the apoptosis in the cells treated with FUDR and IFN-γ. Approximately one third of the population of treated L929 cells showed protection against viral apoptosis compared to virus infected control at 12 hrs. post-infection. No difference was observed at 6 hrs. post- infection. RT-PCR analysis was conducted at 6, 10 and 16 hrs. post infection with HSV-1 at 2 multiplicity of infection to detect expression of ICP0 (infected cell protein) and LAT (Latency associated transcript) viral transcripts. LAT expression was observed at 16 hrs. in the infected control. Immuno-staining was used to detect HSV-1 ICP0 protein in treated L929 cells and virus infected control. A significant difference was observed, with higher expression of ICP0 in virus infected control than in cells treated with FUDR and IFNγ. Image J was used to merge images of actin stained and ICP0 stained cells. In these asynchronous fibroblast cultures treated with FUDR about 5- 10% cells replicate in the presence of FUDR. Four percent of the cells in the FUDR treated population showed ICP0 staining, as expected. A quiescent infection of HSV-1 was established in L929 cells treated with the mitotic inhibitor and maintained in a medium supplemented with IFN-γ. LAT was not detected in treated cells.
Department or Program
Microbiology and Immunology
Year Degree Awarded
Copyright 2012, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.