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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >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
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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

机译：上下文周围的加工位点是决定病毒蛋白酶中HIV-1 Gag和Gag-Pro-Pol多蛋白前体中加工位点亚型的裂解率的关键

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摘要

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), SP1ucleocapsid (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.
机译：HIV-1蛋白酶（PR）对1型人类免疫缺陷病毒（HIV-1）Gag和Gag-Pro-Pol多蛋白的加工对于产生感染性颗粒至关重要。但是，尚不清楚对决定这些基板的处理速率的决定因素。我们通过利用一种新型的蛋白酶测定法研究了底物背景对加工的影响，在该方法中，含有HIV-1基质（MA）和衣壳N末端结构域（CA）的底物被FlAsH（荧光素砷发夹）试剂标记。当将Gag和Gag-Pro-Pol多蛋白内的七个切割位点置于MA / CA位点时，与先前报道的自然背景下的切割位点相比，切割率发生了巨大变化。加工速率对三个位点的影响最大：CA /间隔肽1（SP1）（增加约10倍），SP1 /核衣壳（NC）（减少约10-30倍）和SP2 / p6（≈减少30倍）。两种耐多药（MDR）PR变体之一极大地改变了加工速率的模式。 Pro-Pol区域内的切割位点以上下文无关的方式切割，提示这些位点序列本身是决定速率的因素。此外，由SP1 / NC P4-P1和MA / CA P1'-P4'残基（ATIM↓PIVQ）组成的嵌合体废除了野生型和MDR蛋白酶的加工，而由MA / CA P4-P1和SP1 / NC P1'–4'（SQNY↓IQKG）仅被一种MDR蛋白酶切割。这些结果表明，复杂的底物相互作用既超出酶的活性位点，也贯穿易裂键，均有助于确定HIV-1 PR的加工速率。

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