Mapping of the CXCR4 Binding Site within Variable Region 3 of the Feline Immunodeficiency Virus Surface Glycoprotein

Feline immunodeficiency virus (FIV) infection in cats is considered a valuable small-animal model of human immunodeficiency virus (HIV) vaccination strategies and drug development, as it is the only nonprimate lentivirus that causes a loss of CD4+ T cells followed by an AIDS-like disease in its natural host. Although FIV primarily targets CD4+ T cells (9, 12, 20), it uses CD134 instead of CD4 as its primary binding receptor (12, 40). CD134 is a member of the tumor necrosis factor receptor superfamily and is specifically expressed on activated CD4+ T cells, where it induces antiapoptotic signals to allow for survival and proliferation late in T-cell responses (7). The direct correlation between CD134 upregulation and the preferential targeting of FIV for activated T cells explains the FIV tropism for these cells (12). Similar to T-cell-tropic HIV type 1 (HIV-1), FIV utilizes CXC chemokine receptor 4 (CXCR4) as an entry receptor for infection (48, 50), after interaction with a primary binding receptor (14). Also, the overall genetic structure of FIV is similar to that of HIV (33, 37, 45), and the FIV env gene encodes a surface glycoprotein (SU or gp95) and a transmembrane protein (TM or gp41). The SU protein exhibits considerable amino acid sequence variation, with five consensus major variable (V) regions (V1 to V5) (36, 37). Of these domains, the V3 loop has been identified as a major immunogenic domain in SU by epitope mapping with sera from infected cats (3, 18, 29, 34). Studies of FIV-PPR interactions with CD134 have identified specific amino acids involved in SU binding in the outermost domain 1 of CD134 (11). Recent studies have indicated the involvement of additional residues in domain 2 in binding of certain other strains of FIV (49). For SU interaction with its entry receptor, CXCR4, involvement of the second extracellular loop of CXCR4 has been reported (5, 47). However, the domains of SU involved in direct interaction with CXCR4 have not been identified. The purpose of this study was to define the domains of SU that are crucial for the interaction with CXCR4 in order to better understand the mechanism of FIV entry and infection. Analysis of genetic variations in SU proteins of FIV, simian immunodeficiency virus, and HIV-1 has revealed analogies in the locations and distribution of the variable domains V3, V4, and V5 (35). A structural model of FIV SU identified these variable domains as potential exposed loop segments (36). A segment, denoted loop II (L2), in the N-terminal part of FIV SU has also been predicted to fold as an exposed loop (36). As a step toward a better understanding of the molecular interaction between FIV and its target cells, we created SU mutants with deletions of L2, V3, V4, or V5. These mutants were used together with a panel of SU-specific monoclonal antibodies (MAbs) in SU-CXCR4 binding studies. Using nested V3 peptides and site-directed mutagenesis, the CXCR4 binding region of SU was further localized to a discrete stretch of nine amino acids at the predicted tip of the V3 loop, with critical involvement of serine 393 and tryptophan 394 to facilitate binding and virus entry into the target cell.