A Continuous Fluorometric Assay for the Feline Immunodeficiency Virus Protease

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Section: Discussionsupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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

Lin

Beck

Morris

et al. 2003

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“…Some substrates, such as the matrix-capsid (MA/ CA) junction of HIV-1 GAG, are cleaved by both enzymes, but the actual cleavage site is shifted, a finding indicative of distinctions in substrate binding within the S4-S4Ј binding pocket of each enzyme (17). Analysis of cleavage by HIV-1 PR of a fluorescent substrate designed for FIV PR [Arg-Ala-LeuThr-Lys-(Abz)-Val-Gln2Phe (NO 2 )-Phe-Val-Gln-Ser-LysGly-Arg-NH 2 (9)] revealed that this substrate is cleaved by HIV-1 PR although Ͼ100 times less efficiently than it cleaves an HIV-specific fluorescent substrate ( Table 2). The K m for HIV-1 PR on the FIV substrate is only fourfold higher than the K m for cleavage against the HIV-1 substrate.…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage of fluorescent substrates designed for HIV-1 PR, Abz-Thr-Ile-Nle2 Phe(NO 2 )-Gln-Arg-NH 2 (26), and FIV PR, NH 2 -Arg-Ala-Leu-Thr-Lys(Abz)-Val-Gln2Phe(NO 2 )-Val-Gln-Ser-Lys-Gly-Arg-NH 2 (9), was assessed at pH 5.6 in 0.2 M NaCl-0.1 M MES at 37°C. FIV PR at 368 nM or HIV-1 PR at 297 nM was used in the reactions (see Table 2).…”

Section: Methodsmentioning

confidence: 99%

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

Beck

Lin

Elder

2001

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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.

“…The activity of FIV PR was assayed in 50 mM sodium citrate-100 mM phosphate buffer (pH 5.25) with 200 mM NaCl and 1 mM dithiothreitol at 37°C using 50 M fluorogenic substrate A-L-T-(2-amino benzoic) K-V-Q/(p-NO 2 ) F-V-Q-S-K-G as described previously (18). The sensitivities of chimeric 6s, 6s-98H, and 6s-98S PRs to the inhibitors DRV, LPV, and TL-3 were measured.…”

Section: Pr Assay and Ic 50 S Of Inhibitorsmentioning

confidence: 99%

Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

Lin¹,

Torbett

Elder³

2010

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Feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteases (PRs)share only 23% amino acid identity and exhibit distinct specificities yet have very similar 3-dimensional structures. Chimeric PRs in which HIV residues were substituted in structurally equivalent positions in FIV PR were prepared in order to study the molecular basis of PR specificity. Previous in vitro analyses showed that such substitutions dramatically altered the inhibitor specificity of mutant PRs but changed the rate and specificity of Gag cleavage so that chimeric FIVs were not infectious. Chimeric PRs encoding combinations of the I37V, N55M, M56I, V59I, L97T, I98P, Q99V, and P100N mutations were cloned into FIV Gag-Pol, and those constructs that best approximated the temporal cleavage pattern generated by wild-type FIV PR, while maintaining HIV-like inhibitor specificity, were selected. Two mutations, M56I and L97T, were intolerant to change and caused inefficient cleavage at NC-p2. However, a mutant PR with six substitutions (I37V, N55M, V59I, I98P, Q99V, and P100N) was selected and placed in the context of full-length FIV-34TF10. This virus, termed YCL6, had low-level infectivity ex vivo, and after passage, progeny that exhibited a higher growth rate emerged. The residue at the position of one of the six mutations, I98P, further mutated on passage to either P98H or P98S. Both PRs were sensitive to the HIV-1 PR inhibitors lopinavir (LPV) and darunavir (DRV), as well as to the broad-based inhibitor TL-3, with 50% inhibitory concentrations (IC 50 ) of 30 to 40 nM, consistent with ex vivo results obtained using mutant FIVs. The chimeras offer an infectivity system with which to screen compounds for potential as broad-based PR inhibitors, define structural parameters that dictate specificity, and investigate pathways for drug resistance development.