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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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AIDS Molecular and Cellular Biology Study Section (AMCB)
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Miller, Roger H
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Scripps Research Institute
La Jolla
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Hu, Qiong-Ying; Fink, Elizabeth; Grant, Chris K et al. (2014) Selective interaction of heparin with the variable region 3 within surface glycoprotein of laboratory-adapted feline immunodeficiency virus. PLoS One 9:e115252
Troyer, Ryan M; Thompson, Jesse; Elder, John H et al. (2013) Accessory genes confer a high replication rate to virulent feline immunodeficiency virus. J Virol 87:7940-51
Wood, Britta A; Carver, Scott; Troyer, Ryan M et al. (2013) Domestic cat microsphere immunoassays: detection of antibodies during feline immunodeficiency virus infection. J Immunol Methods 396:74-86
Hu, Qiong-Ying; Fink, Elizabeth; Elder, John H (2012) Mapping of Receptor Binding Interactions with the FIV surface Glycoprotein (SU); Implications Regarding Immune surveillance and cellular Targets of Infection. Retrovirology (Auckl) 2012:1-11
Breuer, Sebastian; Sepulveda, Homero; Chen, Yu et al. (2011) A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease. Biochemistry 50:4371-81
Miller, Craig; Bielefeldt-Ohmann, Helle; MacMillan, Martha et al. (2011) Strain-specific viral distribution and neuropathology of feline immunodeficiency virus. Vet Immunol Immunopathol 143:282-91
Hu, Qiong-Ying; Fink, Elizabeth; Happer, Meaghan et al. (2011) Identification of amino acid residues important for heparan sulfate proteoglycan interaction within variable region 3 of the feline immunodeficiency virus surface glycoprotein. J Virol 85:7108-17
Thompson, Jesse; MacMillan, Martha; Boegler, Karen et al. (2011) Pathogenicity and rapid growth kinetics of feline immunodeficiency virus are linked to 3' elements. PLoS One 6:e24020
Hu, Qiong-Ying; Fink, Elizabeth; Hong, Yang et al. (2010) Fine definition of the CXCR4-binding region on the V3 loop of feline immunodeficiency virus surface glycoprotein. PLoS One 5:e10689
Elder, John H; Lin, Ying-Chuan; Fink, Elizabeth et al. (2010) Feline immunodeficiency virus (FIV) as a model for study of lentivirus infections: parallels with HIV. Curr HIV Res 8:73-80

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