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

ABSTRACT We used feline immunodeficiency virus (FIV) protease (PR) as a mutational framework to define determinants for the observed substrate and inhibitor specificity distinctions between FIV and human immunodeficiency virus (HIV) PRs. Multiple-substitution mutants were constructed by replacing the residues in and around the active site of FIV PR with the structurally equivalent residues of HIV-1 PR. Mutants included combinations of three critical regions (FIV numbering, with equivalent HIV numbering in superscript): I3732V in the active core region; N5546M, M5647I, and V5950I in the flap region; and L9780T, I9881P, Q9982V, P10083N, and L10184I in the 90s loop region. Significant alterations in specificity were observed, consistent with the involvement of these residues in determining the substrate-inhibitor specificity distinctions between FIV and HIV PRs. Two previously identified residues, I35 and I57 of FIV PR, were intolerant to substitution and yielded inactive PRs. Therefore, we attempted to recover the activity by introducing secondary mutations. The addition of G6253F and K6354I, located at the top of the flap and outside the active site, compensated for the activity lost in the I5748G substitution mutants. An additional two substitutions, D10588N and N8874T, facilitated recovery of activity in mutants that included the I3530D substitution. Determination of Ki values of potent HIV-1 PR inhibitors against these mutants showed that inhibitor specificity paralleled that of HIV-1 PR. The findings indicate that maintenance of both substrate and inhibitor specificity is a function of interactions between residues both inside and outside the active site. Thus, mutations apparently peripheral to the active site can have a dramatic influence on inhibitor efficacy.

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