Host Cell Determinants and Retroviral Infection

Indiana Univ-Purdue Univ At Indianapolis, Indianapolis IN

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Abstract

DESCRIPTION (Provided by applicant): The human T cell leukemia virus type 1 (HTLV-1) preferentially infects and transforms CD4+ T lymphocytes in vivo and in vitro. The molecular basis underlying this cell tropism is not understood. A number of candidate molecules have been proposed as potential receptors, however, none of them fulfils the requirements for an obligate viral receptor. The overall goal of this application is to determine the host cell molecules involved in HTLV-1 infection. The first hypothesis to be tested is that HTLV-1 utilizes CD4 for entry into susceptible T lymphocytes. Clinical support for this hypothesis is evident in the preferential infection and transformation of CD4+ T cells in the majority of infected patients. We have recently produced preliminary data to support this hypothesis. By using a novel vaccinia-based reporter gene activation assay developed in our laboratory, we are the first to demonstrate that CD4 expression renders murine cells susceptible for HTLV-1 envelope (Env)mediated cell fusion. The second hypothesis to be tested is that, in addition to CD4, a "coreceptor" is required for the preferential infection of CD4+ T lymphocytes in vivo. This hypothesis is based on preliminary data generated in our laboratory showing that transfection of a HeLa cell cDNA library into NIH-3T3 cells can render them fusogenic with HTLV-1Env and that co-expression of CD4 resulted in ten fold increase in the cell fusion signal. Our preliminary data relied largely on a cell fusion assay that measures the fusogenic potential of Env. The purpose of this application is to obtain further evidence for the role of CD4 in infectivity assays and identify the putative HTLV-1 "coreceptor." The specific aims of this proposal are: 1) Analyze the role of CD4 and CD8 in HTLV-1 infection. 2) Determine the identity of the HTLV- 1 "coreceptor." The first specific aim will utilize HTLV- 1 pseudotyped viruses for infection of a receptor-negative cell line to validate the role of CD4 and correlate cell fusion data with viral infectivity. The second specific aim will utilize a novel functional cDNA screening strategy previously used to isolate the HIV coreceptors. Unlike previously used methods, the proposed screening approach makes no assumptions about the mode of action of the fusion coreceptor, except that it allows a receptor-negative cell type to undergo fusion with cells expressing the HTLV- 1 Env. The experimental methods will utilize procedures and reagents previously utilized by the investigator during his published description of the HIV coreceptors.