Selective interaction of heparin with the variable region 3 within surface glycoprotein of laboratory-adapted feline immunodeficiency virus

Abstract

Heparan sulfate proteoglycans (HSPG) can act as binding receptors for certain laboratory-adapted (TCA) strains of feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV). Heparin, a soluble heparin sulfate (HS), can inhibit TCA HIV and FIV entry mediated by HSPG interaction in vitro. In the present study, we further determined the selective interaction of heparin with the V3 loop of TCA of FIV. Our current results indicate that heparin selectively inhibits infection by TCA strains, but not for field isolates (FS). Heparin also specifically interferes with TCA surface glycoprotein (SU) binding to CXCR4, by interactions with HSPG binding sites on the V3 loop of the FIV envelope protein. Peptides representing either the N- or C-terminal side of the V3 loop and containing HSPG binding sites were able to compete away the heparin block of TCA SU binding to CXCR4. Heparin does not interfere with the interaction of SU with anti-V3 antibodies that target the CXCR4 binding region or with the interaction between FS FIV and anti-V3 antibodies since FS SU has no HSPG binding sites within the HSPG binding region. Our data show that heparin blocks TCA FIV infection or entry not only through its competition of HSPG on the cell surface interaction with SU, but also by its interference with CXCR4 binding to SU. These studies aid in the design and development of heparin derivatives or analogues that can inhibit steps in virus infection and are informative regarding the HSPG/SU interaction.

Figure 1. Selective inhibition of TCA FIV infectivity by heparin.

(A) Productive infection assay of FIV-PPRcr inhibited by heparin in G355-5 and Gfox cells. (B) Productive infection assay of FIV-34TF10 in G355-5 and FIV-PPR in Gfox cells inhibited by heparin. Virus growth in cells was evaluated as CPM by a reverse transcriptase activity assay over time. Heparin was used at 20 µg/ml. Results are means and standard deviations (SD) for three independent determinations. ***p<0.001; **p<0.01; *p<0.05; as compared to the untreated control group, (C). Effect of heparin on FIV TCA entering G355-5 cells and FS entering Gfox cells. Entry assay were performed in the presence or absence of heparin at indicated concentrations. Values are inhibition percentage calculated as described in “Materials and Methods”. Results are means and standard deviations (SD) for three independent determinations. ***p<0.001; **p<0.01; *p<0.05; as compared to the FIV TCA groups.

Figure 2. Heparin selectively interferes with TCA SU/CXCR4 interactions.

(A) Effect of heparin on TCA or FS SUs-Fc binding to 3201 cells (feline CXCR4) and SupT1 cells (human CXCR4). Heparin was used as 20 µg/ml. Values are inhibition percentage calculated as described in “Materials and Methods”. Results are means and SD for four independent determinations. (B) Heparin interferes with PPRcr or PPR SU-Fc binding to CXCR4 at the indicated concentrations. Values are inhibition percentage. Results are means and SD for three independent determinations. ***p<0.001; **p<0.01; *p<0.05; as compared to the PPR group at the same concentration. (C) Heparin interferes with 34TF10 SU binding to CXCR4. 34 SU-Fc and Fc-34 SU were treated with heparin (20 µg/ml). The top panel and bottom panel represent 3201 cells and SupT1 cells, respectively. Results represent one of three independent experiments.

Figure 3. Sequence alignment of the V3 loop of 34TF10, PPRcr, PPR, and C36 surface glycoprotein.

Blue indicates the CXCR4 binding region, and orange indicates the HSPG binding region. Both regions are indicated in bold.

Figure 4. Effect of heparin on PPR mutants binding to CXCR4.

Panel A and B represent 3201 cells and SupT1 cells, respectively. Heparin was used at 20 µg/ml. Values are inhibition percentage calculated as described in “Materials and Methods”. Results are means and standard deviations for triplicate determinations.

Figure 5. V3 peptide affects heparin interference with TCA FIV SU binding to CXCR4.

Panel A and B represent PPRcr and 34TF10 SU-Fc binding to 3201 cells, respectively. All peptides were used at 50 µg/ml as final concentration. Heparin was used at 20 µg/ml. Values are inhibition percentage calculated as described in “Materials and Methods”. Results are means and SD for three independent determinations. ***p<0.001; **p<0.01; *p<0.05; as compared to the heparin treatment group without peptides.