Background:  The identification of new anti-HIV-1 drugs targeting novel sites of action remains a high priority in this era of drug-resistant viruses. One such new drug is PA-457, which potently inhibits both wild type HIV-1 and isolates resistant to current anti-retrovirals. PA-457 disrupts virus maturation, a process essential for virus infectivity. Virus maturation requires the sequential proteolytic cleavage of Gag to matrix, capsid (CA), nucleocapsid, and p6 domains, and spacer peptides SP1 and SP2. PA-457 disrupts the cleavage of CA-SP1 to CA.

Methods:  Viral resistance to PA-457 was examined in vitro and in vivo. PA-457-resistant viral variants, in vitro, were selected by serial passage at a suboptimal drug concentration. We analyzed in vivo, gag sequences from patient samples collected from a 10-day, multi-dose PA-457 monotherapy study.

Results:  The in vitro selection identified 5 amino acid changes that independently confer PA-457 resistance. At the extreme C-terminus of CA, 3 substitutions are located (H226Y, L231M, and L231F) and 2 were identified at the first and third residues of SP1 (A1V and A3V). Mutations H226Y, L231M, L231F, and A1V had little effect on viral replication kinetics in the absence of drug or at various drug concentrations. The A3V change greatly decreased viral fitness in the absence of drug and at a suboptimal drug concentration. Interestingly, A3V fitness increased at a high drug concentration. Electron microscopy revealed that, in the absence of drug, A3V-infected cells released virions with an immature morphology. In the presence of a high drug concentration, A3V-infected cells produced virions with condensed cores. The replication defect imposed by A3V was reversed by a second-site change in CA (G225S). The A3V/G225S mutant was highly fit and fully drug resistant. None of the changes in vivo that were associated with resistance in vitro were observed by population sequencing of patient samples collected during, or following completion of, dosing.

Conclusions:  The clustering of PA-457-resistance-conferring mutations at the CA/SP1 junction confirms that this region is the major target of PA-457 activity. The drug dependence observed for A3V suggests multiple distinct mechanisms of resistance. Resistance to PA-457 was not detected in vivo during a 10-day, multi-dose study using standard genotyping methods. Interestingly, all the amino acid positions to which PA-457 resistance maps in vitro are highly conserved among HIV-1 isolates, suggesting that there may be a fitness cost to PA-457 resistance.