Anti-FIV Glycoprotein 95 Monoclonal antibody, Clone TV3-4

Heparan sulfate proteoglycans (HSPGs) act as binding receptors or attachment factors for the viral envelope of many viruses, including strains of HIV and feline immunodeficiency virus (FIV). The FIV gp95 glycoprotein (SU) from laboratory-adapted strains (tissue culture adapted [TCA]) such as FIV-34TF10 can bind to HSPG, whereas SU from field strains (FS) such as FIV-PPR cannot. Previous studies indicate that SU-HSPG interactions occur within the V3 loop. We utilized a series of nested V3 peptides to further map the HSPG binding sites and found that both sides of the predicted V3 loop stem were critical for the binding but not the CXCR4 binding domain near the predicted tip of the V3 loop. Neutralization assays for TCA strain entry using the same set of V3 peptides showed that peptides targeting CXCR4 or HSPG binding sites can block infection, supporting the V3 loop as a critical neutralization target. Site-directed mutagenesis identified two highly conserved arginines, R379 and R389, on the N-terminal side of the V3 stem as critical for the contact between SU and HSPG. Residues K407, K409, K410, and K412 on the C-terminal side of the V3 stem form a second nonconserved domain necessary for HSPG binding, consistent with the observed specificity distinctions with FS FIV. Our findings discriminate structural determinants important for HSPG and CXCR4 binding by FIV SU and thus further define the importance of the V3 loop for virus entry and infection.

The processing and transport of the envelope glycoprotein complex of feline immunodeficiency virus (FIV) in the persistently infected Crandell feline kidney (CRFK) cell line were investigated. Pulse-chase analyses revealed that the glycoprotein is synthesized as a precursor with an M(r) of 145,000 (gp145) and is quickly trimmed to a molecule with an M(r) of 130,000 (gp130). Treatment of gp130 with endoglycosidase H (endo H) resulted in a protein with an M(r) of 75,000, indicating that nearly half the weight of the gp130 precursor consists of endo H-sensitive glycans during biosynthesis. Chase periods of up to 8 h revealed intermediates during the further processing of this glycoprotein precursor. Initially, two minor protein species with apparent M(r)s of 100,000 and 90,000 were detected along with gp130. At later chase times these two species appeared to migrate as a single dominant species with an M(r) of 95,000 (gp95). Concomitant with the appearance of gp95 was another protein with an M(r) of approximately 40,000 (gp40). Chase periors of up to 8 h revealed that approximately half of the precursor was processed into the gp95 complex with 4 h. gp95 was efficiently transported from the cell into the culture medium by 1 to 2 h after labeling, whereas gp40 was not observed to be released from infected CRFK cells. Analysis of the processing in the presence of monensin, castanospermine, and swainsonine also suggests the existence of these intermediates in the processing of this lentivirus glycoprotein. As with human immunodeficiency virus, virus produced in the presence of glucosidase inhibitors had reduced infectivity for T-lymphocyte cultures.