Background:  We previously demonstrated that the betulinic acid derivative, PA-457, potently inhibits HIV-1 replication by targeting a late step in Gag processing. Specifically, PA-457 blocks the conversion of the capsid precursor (CA-SP1, p25) to mature capsid protein (CA, p24) resulting in the release of immature, non-infectious viral particles. SP1 is a small spacer peptide (13- to 19-amino acid residues) that separates the CA and NC domains in the Gag polyprotein precursor. In vitro selection experiments indicate that determinants of PA-457 activity reside in the region proximal to the Gag CA-SP1 cleavage site. The focus of the current work is to fully characterize the Gag determinants of PA-457 activity.

Methods:  We generated a panel of viruses containing point deletions spanning the Gag CA-SP1 cleavage domain. The effects of these point deletions on viral particle release, replication kinetics, Gag processing, and infectivity were evaluated. Determinants of PA-457 activity were characterized using those mutants that retained a normal Gag processing profile.  

Results:  Consistent with observations from resistance selection experiments, our current results demonstrate that amino acid residues in both the CA and SP1 domains of Gag are critical to PA-457 activity. Specifically, we found that the insertion of the 6 C-terminal CA residues and the 8 N-terminal SP1 residues from HIV-1 into a PA-457-resistant SIV clone was sufficient to confer compound sensitivity.

Conclusions:  Taken together, these findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and provide strong evidence that this compound specifically targets the CA-SP1 boundary domain of the HIV-1 Gag protein.

Keywords: novel therapeutic; new target; maturation inhibitor