Alteration of substrate and inhibitor specificity of feline immunodeficiency virus protease

Feline immunodeficiency virus (FIV) protease is structurally very similar t o human immunodeficiency virus (HIV) protease but exhibits distinct substra te and inhibitor specificities. We performed mutagenesis of subsite residue s of FIV protease in order to define interactions that dictate this specifi city The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. Th e I37V, N55M, V59I, and Q99V mutants showed a significant increase in activ ity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid j unction peptides compared with wild-type (wt) FIV protease. The I37V: V59I, and Q99V mutants also showed an increase in activity against two rapidly c leaved 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-I matrix/ capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, i ndicating that none of the substitutions were sufficient to change the spec ificity 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 hydro phobic 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 re garding the interactions involved in substrate binding and aid in the devel opment of broad-based inhibitors.