Mutational analysis of a native substrate of the human immunodeficiency virus type 1 proteinase.
Proteolytic processing of the gag/pol precursor by the human immunodeficiency virus type 1 proteinase is essential for the production of infectious viral particles. Although the sites of virus-specific cleavages have been determined, the primary amino acid sequences surrounding these sites are heterogeneous and the determinants that direct the cleavage specificity exhibited by human immunodeficiency virus type 1 proteinase remain largely undefined. We performed mutational analysis of the Tyr/Pro site, which produces the amino terminus of the viral capsid protein, and the Phe/Pro site, which produces the amino terminus of the proteinase. Mutations were made in a clone encoding a frameshift mutation that results in the expression of equimolar amounts of the substrate and proteinase in the form of a truncated gag/pol precursor. After single-amino-acid substitutions were made, their effects on proteolytic processing were examined by in vitro transcription and in vitro translation of the synthetic mRNA; translation products were then processed by exogenously added purified proteinase. Single-amino-acid substitutions yielded both substrates which were processed with wild-type efficiency and substrates on which processing was impaired. At the Tyr/Pro site in gag, processing was severely inhibited by substitutions within the P4, P2, P1, and P2' positions. The Phe/Pro site in pol, however, demonstrated far greater tolerance to amino acid substitution. These data suggest that the primary amino acid sequence around a scissile bond is more critical for cleavage of the Tyr/Pro site than the Phe/Pro site.
Department of Microbiology, State University of New York, Stony Brook 11794.
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D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000235:genetics* / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics*; D015684:Gene Products, pol / Q000378:metabolism*; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D015684:Gene Products, pol; D010450:Endopeptidases; D016333:HIV Protease