Mutational analysis of a native substrate of the HIV-1 proteinase.
The purpose of this study was to define further the determinants of substrate specificity of HIV-1 PR. Rather than using small peptides, we used an in vitro system which permitted us to evaluate the effect of a mutated site within the context of its natural precursor. We made single-amino-acid substitutions around two sites which are processed by the HIV-1 PR. The Tyr/Pro site within gag appears to encode highly specific determinants which direct proteinase processing between MA and CA. The Phe/Pro site in pol, however, appears to be far more tolerant to amino acid substitutions, as none of our single-amino-acid substitutions blocked cleavage at or around this site. The increased tolerance of the Phe/Pro site may indicate that at this site, structural features are more important determinants of cleavage than primary amino acid sequence. We have shown that sequences outside of those encoding mature PR can inhibit proteolytic processing in this system. By preventing PR from cleaving itself from the polyprotein prematurely, p6* sequences would regulate morphogenesis and infectious particle formation. Late in infection, when the protein concentration of gag and gag/pol polyproteins at the cell surface becomes very high, cooperative protein-protein interactions may cause alterations of a p6*-PR interaction, relieving repression and permitting autocatalysis.
Partin
K
K
Department of Microbiology, State University of New York, Stony Brook 11794-8621.
Wimmer
E
E
Carter
C
C
D000595:Amino Acid Sequence; D001665:Binding Sites; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism; D006678:HIV / Q000235:genetics / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010435:Pepsinogens / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D015685:Fusion Proteins, gag-pol; D010435:Pepsinogens; D016333:HIV Protease