V3 loop peptides from HIV-1 strain MN have different polyanion binding specificities than the DBP subdomain 1 HBM peptide

The heparin affinities of hbs-wt peptide, the cyclic V3 loop peptide of HIV-1 strain MN, and RANTES were the same when tested in heparin-Sepharose columns, and X4 strains of HIV, PvRII and hbs-wt can be inhibited by the same polyanions, but not chondroitin sulfate. To see if there are differences in the specificity by which the V3 peptides and the hbs-wt peptide bind to heparin, an ELISA based on RANTES binding to BSA-heparin was developed much like the hbs-wt ELISA. The only difference to the hbs-wt ELISA is that RANTES is substituted for the hbs peptide, and detection is through a biotinylated mouse anti-RANTES monoclonal antibody followed by horseradish peroxidase-conjugated streptavidin. The V3 loop peptides and hbs peptides were added at different concentrations to compete with RANTES at a fixed 5 nM. The linear and cyclic V3 loop peptides of strain MN that had significant binding on heparin-Sepharose were able to compete with RANTES binding to BSA-heparin in a dose-dependent manner, but the hbs-wt peptide was not.

The subdomain 1 HBM has a conserved role in the DBP protein family for binding to diverse receptors, but only members of the family that bind to DARC are inhibited by polyanions

Studies by Ranjan and Chitnis have identified a site in PvRII in the C-terminal flanking region to the DBP V3-like peptide, between C4 and C7, that contain residues necessary for DARC binding [27]. This study also showed that the C1-C4 region of the P. knowlesi beta protein, a member of the DBP family that does not bind to DARC, was capable of substituting for the P. vivax C1-C4. Upon closer inspection, the consensus heparin binding motif is well conserved in the DBP family, with great similarity between proteins that bind different receptors. The P. knowlesi alpha and gamma proteins have an identical consensus heparin binding site, but only alpha binds to DARC. To see if the consensus heparin binding motif may play a similar role in the binding proteins of other members of the DBP family, the same three alanine substitutions found in pv22KARA were introduced in our previous report by site directed mutagenesis into the plasmids pHKADR22, pHKBDR22, and pHKGDR22. This yielded the constructs pkalphaKARA, pkbetaKARA, and pkgammaKARA, which contain the K221, R224, and R227 alanine substitution in the P. knowlesi alpha, beta, and gamma genes, respectively. All three of these mutants failed to bind rhesus erythrocytes when expressed in COS-7 cells, whereas the parent vectors bind very well. When binding of rhesus erythrocytes to the wild type plasmids in COS-7 cells was measured in the presence of polyanions, only the DARC binding alpha protein was inhibited in a dose-dependent manner.