Date of Award

1-4-2010

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Michael Kashgarian MD

Second Advisor

Stacey Efstathiou MD

Abstract

The human gammaherpesviruses EBV and KSHV realize their oncogenic potential during latent infection. The species specificity of the human gammaherpesviruses has hindered the study of latency in animal models. Murine gammaherpesvirus MHV-68 (MHV-68) may be used as a representative gammaherpesvirus for the study of latency. The goal was to establish an in vitro model of MHV-68 latency using replication defective MHV-68. ORF50 has been identified as the major viral trans-activator essential for entry into the lytic replication cycle and necessary and sufficient for reactivation of MHV-68 virus from latency. ORF50 null mutants (A50) can theoretically be used to infect cells in vitro to facilitate an analysis of virus gene expression and episome maintenance during latency. In this project A50 mutants containing the luciferase or green fluorescence protein (GFP) under OW50 promoter control were used to infect a variety of cell types. 3T3 fibroblasts are a permissive cell line and were used for an initial characterization of the ability of A50 MHV-68 to establish latency. B lymphocytes and macrophages are the major reservoirs of persistence in vivo thus the ability of A50 mutants to establish latency in NSO B and RAW macrophage cell lines was explored. Latency was readily established and maintained in 3T3 and RAW cells. The low infectability of NSO B- cells restricted the utility of this cell line in studies of latency. Examination of patterns of lytic and latent transcription in 3T3 and RAW cells coordinately infected with A50 MHV-68 revealed reactivation efficiencies of 40-60%. Following long-term passage A50 exhibited stable transcription of two latency related genes M2 and ORF73, with episomal maintenance of the viral genome, in the absence of contaminating lytic infection. The results demonstrate the utility of A50 mutants for studies of gammaherpesvirus latency in vivo.

Share

COinS