title: A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.
abstract: HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Counts
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: P Shing
authors.initials: PS
authors.lastname: Ho
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America; Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Maureen J
authors.initials: MJ
authors.lastname: Donlin
authors.affiliation: Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: John E
authors.initials: JE
authors.lastname: Tavis
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Chaoping
authors.initials: C
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000069454:Darunavir / Q000494:pharmacology; D057809:HEK293 Cells; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D054301:gag Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; D000069454:Darunavir