title: Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease.
abstract: Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.
authors.affiliation: Department of Biochemistry and Biophysics, and the University of North Carolina Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Sook-Kyung
authors.initials: SK
authors.lastname: Lee
authors.forename: Marc
authors.initials: M
authors.lastname: Potempa
authors.forename: Madhavi
authors.initials: M
authors.lastname: Kolli
authors.forename: Ayşegül
authors.initials: A
authors.lastname: Özen
authors.forename: Celia A
authors.initials: CA
authors.lastname: Schiffer
authors.forename: Ronald
authors.initials: R
authors.lastname: Swanstrom
mesh_terms: D000595:Amino Acid Sequence; D016325:HIV Long Terminal Repeat / Q000502:physiology; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D013379:Substrate Specificity / Q000502:physiology; D014771:Virion / Q000201:enzymology; D019065:Virus Assembly / Q000502:physiology; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1