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

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date


Document Type



Amino Acid Sequence; HIV Long Terminal Repeat; HIV Protease; HIV-1; Substrate Specificity; Virion; Virus Assembly; gag Gene Products, Human Immunodeficiency Virus; pol Gene Products, Human Immunodeficiency Virus


Biochemistry, Biophysics, and Structural Biology | Molecular Biology


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) ( approximately 10-fold increase), SP1/nucleocapsid (NC) ( approximately 10-30-fold decrease), and SP2/p6 ( approximately 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 downward arrowPIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY downward arrowIQKG) 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.

DOI of Published Version



J Biol Chem. 2012 Apr 13;287(16):13279-90. Epub 2012 Feb 13. Link to article on publisher's site

Journal/Book/Conference Title

The Journal of biological chemistry


Co-author Aysegul Ozen is a student in the Biochemistry & Molecular Pharmacology program in the Graduate School of Biomedical Sciences (GSBS) at UMass Medical School.

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