Mapping of Receptor Binding Interactions with the FIV Surface Glycoprotein (SU); Implications Regarding Immune Surveillance and Cellular Targets of Infection

Abstract

Similar to HIV, FIV uses a two-receptor mechanism to infect CD4+ T cells, the primary target cells in the cat. The T cell activation marker, CD134, serves as a primary binding receptor similar to the role of CD4 for HIV and facilitates interaction with the entry receptor, CXCR4. Heparan sulfate proteoglycans (HSPG) can also act as binding receptors for certain tissue culture adapted FIV and HIV isolates. In the present study, we employed site-directed mutagenesis to investigate the importance of specific residues on the FIV envelope for CD134 and HSPG interactions. We show that certain mutations that disrupt CD134 interactions facilitate HSPG binding by FIV-PPR. In particular, an E407K mutation at the base of the V3 loop knocks out CD134 binding; enhances HSPG binding; and in combination with additional Env mutations E656K and V817I increases entry into CD134−, CXCR4+ target cells by greater than 80-fold over wild type FIV-PPR. The CD134-independent mutant, termed FIV-PPRcr, exhibits a broadened host cell range, but also becomes readily susceptible to CD134-dependent neutralizing monoclonal antibodies. The findings are consistent with the notion that FIV-PPRcr Env has an “open” conformation that readily associates with CXCR4 directly, similar to wild type FIV-PPR Env after CD134 binding. The findings highlight the utility of a two-receptor mechanism that allows FIV V3 residues critical for CXCR4 binding to remain cryptic until reaction occurs with the primary binding receptor, thus thwarting immune surveillance.

Selective Interaction of Heparin with the Variable Region 3 within Surface Glycoprotein of Laboratory-Adapted Feline Immunodeficiency Virus

“…To determine whether the inhibition of viral infectivity by heparin correlated with the ability of heparin to selectively interfere with FIV surface glycoprotein (SU) interaction with receptors, we employed FACS analyses to assess the influence on receptor binding to CD134, HSPG and CXCR4 using specific target cells bearing each receptor. As previously reported [38] , heparin can inhibit FIV TCA SU-Fc binding to CrFK cells but cannot inhibit FIV FS SU-Fc binding to Gfox cells. Which suggests that FIV TCA display significantly more sensitivity to heparin inhibition than does FS, at least partially due to its complete interference with TCA SU binding to HSPG and its weak or undetectable interference with FS SU binding to CD134 by heparin.…”

“…Micro-RT activity assay was performed as previously described [38] Briefly, 50 µl of cell-free supernatant together with 10 µl of lysis buffer (0.75 M KCl, 20 mM dithiothreitol, 0.5% Triton X−100) was incubated at room temperature for 10 minutes. Then, 40 µl of a mixture containing 125 mM Tris-HCl (pH 8.1), 12.5 mM MgCl 2 , 1.25 µg poly(rA)-poly(dT) 12–18 (Amersham Biosciences, Piscataway, NJ) and 1.25 µCi of [ 3 H]dTTP (DuPont, Boston, MA) was added to the sample followed by 2 h of incubation at 37°C.…”

“…Then, 40 µl of a mixture containing 125 mM Tris-HCl (pH 8.1), 12.5 mM MgCl 2 , 1.25 µg poly(rA)-poly(dT) 12–18 (Amersham Biosciences, Piscataway, NJ) and 1.25 µCi of [ 3 H]dTTP (DuPont, Boston, MA) was added to the sample followed by 2 h of incubation at 37°C. The measurement of RT activity was previously described [38] .…”

The virus–receptor interaction in the replication of feline immunodeficiency virus (FIV)

“…In vivo , there is a strong selective pressure to restore a glutamate residue at these positions and revert to virulence [ 16 ]. One of the driving forces for this reversion may be escape from neutralising antibodies induced by CD134-binding; a CD134-independent E407K mutant of PPR was readily susceptible to CD134-dependent neutralising antibodies [ 17 ].…”

“…As polyclonal sera elicited in infected cats can be remarkably mono-specific, targeting single determinants in the variable loops of the virus [ 35,36 ], in vivo escape from neutralisation may be sufficient to drive the emergence of CRD2-independent viruses. In doing so, the progeny viruses may gain the ability to spread into additional cellular compartments through a direct (CD134-independent) interaction with CXCR4 [ 17 ].…”