4.6 Article

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

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 287, Issue 16, Pages 13279-13290

Publisher

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.339374

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Funding

  1. National Institutes of Health [P01-GM066524]

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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) (approximate to 10-fold increase), SP1/nucleocapsid (NC) (approximate to 10-30-fold decrease), and SP2/p6 (approximate to 30-fold decrease). One of two multi-drug- 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 down arrow 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 down arrow 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.

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