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.

FIG. 1

(A) Structure-based amino acid sequence alignment of HIV-1 and FIV proteases. The aligned residues surrounding the substrate-binding pocket of the HIV-1, FIV, SIV, EIAV, and RSV proteases are shown in boxes. The sequences of HIV-2 protease in these three regions are identical to those of SIV protease. ∗, catalytic aspartic acid. (B) Protease cleavage sites at the Gag and Gag-Pol polyproteins of FIV and HIV-1. Please note that the P2 of FIV is different from the P2 of HIV-1, although they have the same nomenclature.

FIG. 2

(Top) Structural locations of substituted residues of FIV protease. These residues include I35 and I37 of the active core; Q54, N55, M56, I57, and V59 of the flap region; and I98, Q99, and L101 of the C-terminal region. These residues are shown on one monomer only. (Bottom) Structural locations of equivalent residues of HIV-1 protease. They are D30, V32, K45, M46, I47, G48, I50, P81, V82, and I101, respectively. These corresponding residues of HIV-1 protease were found to be associated with drug resistance.

FIG. 3

(A and B) Mutant FIV proteases showed increased activities relative to the wt when tested against two peptides representing the HIV-1 P2/NC (A) and RT/IN (B) junction peptides. The protease assay is described in Materials and Methods, and reverse-phase HPLC was used to separate and quantify the peaks. For assaying the P2/NC peptide, 300 nM protease and a 20-min incubation were used. For the RT/IN peptide, 150 nM protease and 5 min of incubation were used. Data are the means plus or minus standard deviation of three independent experiments. (C) The mutant FIV proteases did not show significantly higher activities than the wt when tested against the HIV-1 CA/P2 junction peptide. The assay was done with 300 nM protease and 20 min of incubation. Data are the averages of two independent experiments.

FIG. 4

(A and B) FIV mutant proteases showed increased activities relative to the wt when tested against two rapidly cleaved peptides, GSGIM/FESNL (A) and GSGVF/VEMPL (B), selected by cleavage of a phage display library with HIV-1 protease as described before (2). For assaying these two peptides, 300 nM protease and 20 min of incubation were used. These two peptides were completely cleaved by HIV-1 protease in this assay. Data are the means plus or minus standard deviation of three independent experiments. (C) FIV wt protease showed activity similar to that of the HIV-1 protease when tested against another rapidly cleaved phage peptide, GSGVF/VVNGL. We used 75 nM protease and 5 min of incubation for the assay. Data are the averages of two independent experiments.

FIG. 5

Chemical structures of protease inhibitors used to assay IC₅₀s and their inhibition constant (K_i) against HIV-1 and FIV proteases as described before (22). The interaction between the TL-3 inhibitor and residues in the subsites of FIV protease is also shown based on the described structure (23).

FIG. 6

FIV mutant proteases showed inhibitor specificities more similar to that of the HIV-1 protease. Inhibition of FIV mutant proteases by three protease inhibitors was plotted on different scales due to their different potencies. The IC₅₀s of TL-3 (A), TL-5 (B), and VL-346 (C) are shown.