Hill M K, Hooker C W, Harrich D, Crowe S M, Mak J
AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria 3078, Australia.
J Virol. 2001 Aug;75(15):6835-40. doi: 10.1128/JVI.75.15.6835-6840.2001.
The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.
Gag-Pol在细胞内的运输以及随后整合到人免疫缺陷病毒1型（HIV-1）中的过程仍不清楚。Gag-Pol与Gag由相同的mRNA编码，并通过核糖体移码产生。Gag和Gag-Pol的多聚化是形成传染性病毒颗粒的关键步骤。在本研究中，我们检测了Gag与Gag-Pol之间的相互作用是否在蛋白质翻译过程中启动，以促进Gag-Pol运输到病毒体并随后包装到病毒体中。我们开发了一种条件共转染系统，在该系统中，病毒体形成需要共表达两个基于HIV-1的质粒，一个产生Gag和Gag-Pol，另一个只产生Gag-Pol。Gag-Pol蛋白要么用His表位进行免疫标记，要么在与耐药性相关的逆转录酶中用一个突变（K65R）进行功能标记。评估Gag-Pol的包装情况，以确定整合到病毒体中的Gag-Pol是优先从表达Gag和Gag-Pol的质粒中包装而来，还是可以从任何一个质粒中包装而来。我们的数据表明，从相同mRNA翻译Gag和Gag-Pol对于Gag-Pol多聚蛋白的病毒体包装或病毒功能并不关键。
