Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

Abstract

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3 region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF2VVNGLVK-NH 2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2 position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2 subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1 subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF2VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF⌿(CH 2 NH)VVNGL-NH 2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.

Structural Basis for Distinctions between Substrate and Inhibitor Specificities for Feline Immunodeficiency Virus and Human Immunodeficiency Virus Proteases

“…However, in no case was cleavage as efficient as with HIV-1 PR. The phage peptide contains Glu at P2Ј and thus is not cleaved by wild-type FIV PR (3,4). These results are consistent with the observation that the HIV fluorogenic substrate, containing Gln at P2Ј, is not cleaved by FIV PR.…”

“…We previously performed extensive mutagenesis of residues associated with the S4 to S4Ј subsites of FIV PR in order to attempt to identify residues associated with substrate and inhibitor specificities for FIV and HIV-1 PRs (4,21). Substrate specificity was analyzed by assaying the cleavage efficiency of the mutant PRs on peptides representing FIV and HIV-1 viral cleavage junctions and phage display library peptides which were selected with HIV-1 PR (3).…”

Comprehensive Bioinformatic Analysis of the Specificity of Human Immunodeficiency Virus Type 1 Protease

“…"Cleaved" oligopeptides were those that had been labeled as "cleaved." Three oligopeptides were listed by Beck et al (4) in two slightly different ways, as "Ͻ5% cleavage" and as "not cleaved," which we labeled as uncleaved. These were KGSGVY*QLSALVPK, KGSGGRIN*VALVPK and KSGVF*SVNGLVK, where the scissile bonds are marked with asterisks.…”

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

“…Moreover, the experimentally determined cleavage rates of the cleavable peptides agreed well with the CRM predicted rates on HXB2 and mutated HIV-1 proteases (68% accuracy; RMSEP ¼ 1.01; Figure 1B). Addition of all experimental data to the CRM further increased CRM (Table 3, numbers [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Shown is the predicted versus experimentally determined cleavage rates by HXB2 (red) and mutant HIV-1 proteases, I84V (blue), L90M (magenta), and I84 þ L90M (green).…”

“…The genomic differences among HIV-1 proteases can be as high as 30% and range from 10%-70% within the retroviral protease class [3]. Mutations contributing to viral resistance to antiviral drugs in one particular HIV subtype are found frequently in equivalent positions in the genes of other HIV subtypes or other retroviral proteases [9][10][11][12][13][14]. Still, the roles of specific mutations are only partly understood [5].…”