臺灣博碩士論文加值系統

人類後天免疫不全病毒第一型 (HIV-1)在形成病毒粒子後，必須經由病毒的蛋白酵素 (protease; PR)水解為成熟且具感染力的病毒粒子。蛋白酵素是經由Pol蛋白所衍生的產物，Pol與Gag蛋白以同一條的mRNA為模板被製造出來，在合成Pr55gag發生核糖體往前移位( -1 ribosomal frameshift )跳至Pol 蛋白的open reading frame，使Pol蛋白以一個160 kDa的Gag-Pol聚合蛋白被表現出來。在Pr160gag-pol上的Gag蛋白C端p1與p6已不存在取而代的是另ㄧ個稱為轉架蛋白 ( transframe; p6*; p6pol )。In vitro的研究發現刪除掉p6*可以增強蛋白酵素水解Gag蛋白的能力。因此，認為p6*具有調控蛋白酵素的能力。為了研究p6*對於病毒蛋白酵素活化的影響，我們在Gag-Pol聚蛋白內的p6*區域建構一系列的突變包括刪除p6*，將這些突變的質體與HIV-1 Gag蛋白的表現質體共同轉染293T細胞來觀察病毒粒子的組裝與水解。結果顯示，在Gag-Pol刪除掉p6*對蛋白酵素水解Gag蛋白的能力有些許影響，但如Gag-Pol上刪除Gag或保留完整的p6gag條件下，此時刪除p6*則會降低蛋白酵素水解Gag蛋白能力。而阻斷p6*/PR的切離則明顯降低蛋白酵素水解Gag蛋白的能力，這些實驗的結果暗示p6*的存在也許可以幫助蛋白酵素的活化，如增強Gag-Pol的二聚體化 (dimerization)，但活化中的蛋白酵素必需將p6*從蛋白酵素切離，蛋白酵素才能發揮全面的酵素活性。

The maturation of human immunodeficiency virus particles mediated by viral protease (PR) is not required for virus assembly but is essential for viral infectivity. The PR is encoded by the pol gene, which partially overlaps with the gag open reading frame and is translated as a Gag-Pol polyprotein precursor. Within the Gag-Pol, the C-terminal Gag cleavage product p6gag is truncated and replaced by a transframe peptide referred to as p6* or p6pol. The p6* separates the Gag nucleocapsid (NC) domain from PR. Removal of the p6* can improve HIV Gag processing in vitro,
suggesting that the p6* is involved in the regulation of protease activation. To investigate the contribution of p6* to virus particle maturation, a series of Gag-Pol constructs with various mutations in the p6* was engineered. Effects of the p6* mutations on virus particle assembly and processing were analyzed by coexpressing each of the p6* mutants with an HIV-1 Gag precursor expression plasmid in 293T cells. Analysis indicated that the deletion mutations in p6* markedly affect virus infectivity although Western blot analyses suggested that the deletion mutations have no significant effects on the PR-mediated Gag processing.
However, negative effects of the p6*-deletion mutations on PR-mediated virus particle processing were readily observed when the upstream gag coding sequence had been deleted or an intact C-terminal p6gag was present. Consistent with the previous reports, mutations blocking p6*/PR cleavage significantly affected PR activity. These results suggest that the presence of p6* is required during the process of PR activation; however, the p6* needs to be removed eventually in order to obtain a fully functional PR.

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