Analysis of insertion mutants in distal α9 portion of C-terminal heptad repeat (CHR) of HIV-1 gp41 subunit

Similar to recent JRFL mutants, we have observed the insertion of alanine at the distal α9 of gp41(653+A) severely attenuated membrane fusion while the insertion of glutamine (653+Q) did not. Here we analyzed the mechanism using corresponding synthetic peptides. The both mutant C34 peptides showed less efficient inhibition of membrane fusion even if they were added at the beginning of the coculture of the effector and target cells. The level of the inhibition was similar to that of the wild type C34 added after 30 min of coculture; indicating slow association of mutant C34 peptides with N-terminal heptad region of gp41. Since mixtures of mutant C34 and N36 peptides failed to give an interpretable CD profiles, we tested the longer peptide pairs (C46 and N42) and observed the CD profile indicative of a weak α-helix formation. The melting temperatures for C46-N42 pairs of 653+A, 653+Q and wild type were 56.8°C, 59.8°C, and 96°C, respectively. Our data suggest that the dramatic phenotypic difference in membrane fusion between 653+A and 653+Q (or wild type) cannot be explained by the stability of six-helix bundle (6HB), but the difference in the kinetics of 6HB formation. We also tested other insertions such as E, R, I and L at position 653. Only I and L showed the recovery of the fusion like Q. Our data suggest that the polar nature of glutamine is not a determinant for the phenotypes.