Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

Abstract

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.

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

“…Previous results showed that the inhibitor and substrate specificities of chimeric FIV/HIV PRs could be altered drastically and made more similar to that of HIV-1 PR by introducing multiple substitutions into the active site of FIV PR (36)(37)(38). The altered specificity of chimeric FIV mutants was shown both in in vitro PR assays and in a cell-based Gag-Pol expression system.…”

“…Various combinations of substitutions, including I37 32 V, N55 46 M, V59 50 I, I98 81 P, Q99 82 V, and P100 83 N, were further placed in an FIV PR background and analyzed. Other notable mutations, including I35 30 D, located in the active core, I57 48 G in the flaps, and L101 84 I in the "90s loop," yielded inactive PRs, as determined by the fluorogenic assay in vitro (36,37), and thus were not included in the present study.…”

“…Our approach in studying substrate and inhibitor specificities has been to exchange amino acid residues in and around the active site of FIV PR with structurally equivalent residues of HIV-1 PR. Earlier studies showed that the drug specificity of FIV/HIV-1 chimeric PR could be dramatically changed to HIV-1-like specificity by the substitution of as few as 4 amino acids (I37 32 V, N55 46 M, M56 47 I, and V59 48 I), causing the K i values for the HIV-1 PR inhibitors to drop from the millimolar range down to around 20 nM (36,37). The use of a cell-based FIV Gag-Pol expression system (25) further allowed ex vivo assessment of the processing efficiency and the order of FIV Gag polyprotein processing by chimeric PRs in the context of the natural polyprotein substrate (38).…”

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

“…Because retroviral proteases are inherently dynamic structures that undergo significant structural changes with binding, we described each structurally aligned amino acid of the 61 retroviral proteases by their principal physicochemical properties (i.e., their z-scales z 1 -z 5 ), rather than using the proteins' static 3-D structures (see Materials and Methods, Figure S1, and Table S2) [15,16]. Similarly, we described the retroviral protease substrates by considering the same principal physicochemical properties of every single amino acid of the octapeptide sequence spanning the P 4 to P 4 9 position (see Materials and Methods for details). Protease cleavage rates are dependent on the constituents of the experimental assay (e.g., pH and salt concentrations) [17].…”

“…As such, these proteins are key targets for the design of therapeutic inhibitors [4,5]. To date, the majority of protease inhibitors for treatment of HIV have been peptide mimetics, and most of them were specifically designed against only one of the HIV-1 proteases, namely the HXB2 (''wild-type'') HIV-1 protease [6,7].…”