Most HIV-1-infected individuals with virological failure (VF) on a pharmacologically-boosted protease inhibitor (PI)-containing regimen do not develop PI-resistance protease mutations. One proposed explanation is that mutations in HIV-1 gag or gp41 cytoplasmic domain might also reduce PI susceptibility. In a recent study of paired gag and gp41 sequences from individuals with VF on a PI-containing regimen, we did not identify PI-selected mutations and concluded that if such mutations existed, larger numbers of paired sequences from multiple studies would be needed for their identification. In our study, we performed dN/dS analyses and curated gag and gp41 sequence datasets from more than 400 published studies to generate site-specific amino acid profiles and to identify positions changing during therapy from conserved to uncommon amino acids. The methods used to perform the dN/dS analysis and to generate the curated sequence datasets and amino acid profiles will facilitate the application of consistent methods to a growing body of paired gag and gp41 sequence datasets and expedite the identification of sites under PI-selection pressure.