Alteration of substrate and inhibitor specificity of feline immunodeficiency virus protease.
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.
Lin
Y C
YC
Departments of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Beck
Z
Z
Lee
T
T
Le
V D
VD
Morris
G M
GM
Olson
A J
AJ
Wong
C H
CH
Elder
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002415:Cats; D005456:Fluorescent Dyes / Q000378:metabolism; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D006801:Humans; D016089:Immunodeficiency Virus, Feline / Q000201:enzymology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D012191:Retroviridae Proteins / Q000378:metabolism; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D019151:Peptide Library; D010455:Peptides; D011480:Protease Inhibitors; D012191:Retroviridae Proteins; D054303:HIV Reverse Transcriptase; D016282:Aspartic Acid Endopeptidases; C095555:FIV protease; D016333:HIV Protease