Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease

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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.

Section: Discussionmentioning

confidence: 55%

mentioning

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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“…The V59I substitution would affect P1/P1Ј binding, and the Q99V substitution generates slightly larger S1/S1Ј and S2/S2Ј subsites and would affect P1/ P1Ј and P3/P3Ј binding. The small differences are evident in the conformation of the Phe side chains of TL-3 at the P1/P1Ј positions in the complexes with the V59I and Q99V mutants (23). This correlates with improvement in the K i of TL-3 against these two mutants (21, 23).…”

Section: Discussionmentioning

confidence: 99%

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

Lin¹,

Beck²,

Lee

et al. 2000

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Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2 residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2 might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.

“…All atoms were allowed to move, and energy minimization was performed for 500 iterations of conjugate gradients with both wild-type and the 6x mutant viruses. The starting structure of the HIV-1 protease inhibitor, TL-3, complexed with the wild-type form of HIV-1 protease was taken from the crystal structure (Protein Data Bank accession code 3TLH) (23). The presumed structure of the 6x mutant of HIV-1 protease was constructed by using homology modeling, based on wild-type HIV-1 protease complexed with TL-3 (Protein Data Bank accession code 3TLH).…”

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confidence: 99%

Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3

Bühler¹,

Lin²,

Morris³

et al. 2001

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TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.