The proposed research is for continuation of studies to characterize the feline immunodeficiency virus (FIV) genome, to define mechanisms of entry into target cells and details of the virus life cycle. The ultimate goal of these studies is to understand all aspects of FIV replication, particularly as regards mechanisms shared with HIV and with the purpose of using the feline/FIV model for development of broad-based intervention strategies against lentivirus infections in general. The focus of this grant period is the characterization of envelop/receptor interactions and defining the molecular mechanisms of virus entry into the target cell.
The Specific Aims of the proposal are to 1) Map binding epitopes of a panel of monoclonalantibodies that recognize distinct regions of the FIV envelope glycoprotein. We have produced a panel of 20 monoclonal antibodies that recognize SU of FIV. Four of these antibodies block glycoprotein/receptor interactions and neutralize virus infection in a CD134-independent manner. Mapping of these antibody epitopes, coupled with site-directed mutagenesis and analysis of deletion mutants, will be performed to aid in defining regions of the molecule involved in both CD134 and CXCR4 binding. 2) Carry out Co-crystallization studies of CD134- dependent neutralizing monoclonal antibodies and peptides containing target epitopes. In collaboration with the laboratory of Dr. Ian Wilson, we will perform co-crystallization experiments with synthetic and recombinant peptides and one or more of the neutralizing Mabs to define the local structure around this epitope. Other epitopes that block SU binding to CXCR4 and may or may or may not elicit CD134- dependant neutralization will also be included in these studies, once the epitopes have been mapped in studies under Specific Aim 1;and 3) Analyze the CD134, CXCR4, and HSPG binding properties of a series of deletion constructs lacking specific Env variable regions. All constructs will be prepared as immunoadhesins in CHO cells and analyzed for ability to bind the three receptor types, as assessed by FACS analyses. The latter analyses will be further refined by use of site-directed mutagenesis to map residues critical to each receptor interaction, once minimal receptor binding domains have been identified. Deletion constructs containing minimal binding domains for CD134 will also be utilized in antibody mapping studies under Aim 1 and in crystal trials under Aim 2. We will also assess the influence of envelope deletions on virus infectivity in the context of single round infection by beta-galactosidase-expressing FIV engineered to express each Env mutant. Together, the proposed studies will aid in elucidation of virus/host cell interactions leading to chemokine receptor-mediated entry into the target cell. Understanding the mechanisms of virus entry will aid in development of methods to intervene with virus infection.
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