Wu X, Liu H, Xiao H, Conway J A, Hehl E, Kalpana G V, Prasad V, Kappes J C
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Virol. 1999 Mar;73(3):2126-35. doi: 10.1128/JVI.73.3.2126-2135.1999.
The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.
人类免疫缺陷病毒1型（HIV-1）整合酶蛋白（IN）对于将病毒DNA整合到宿主细胞染色体中至关重要。由于IN作为160 kDa Gag-Pol前体多蛋白的一部分表达并组装到病毒粒子中，并作为成熟的32 kDa蛋白催化感染细胞中前病毒的整合，因此IN中的突变具有多效性，除了整合外，还可能在病毒生命周期的不同阶段影响病毒复制。已观察到IN突变病毒有几种不同的表型，包括病毒粒子形态、蛋白质组成、逆转录、核输入和整合方面的缺陷。由于Gag-Pol的IN结构域中的突变效应并不总是能与成熟IN蛋白中的突变效应区分开来，我们对IN在体内功能的理解仍存在重大差距。为了在复制病毒的背景下直接分析成熟IN蛋白本身的功能，且独立于Gag-Pol进行分析，我们采用了我们实验室开发的一种方法，即将蛋白质作为Vpr或Vpx的融合伴侣通过反式表达掺入HIV病毒粒子中。通过以Vpr-IN融合蛋白的形式反式提供IN，我们的分析首次揭示，成熟IN蛋白对于感染细胞中逆转录的有效起始至关重要，并且该功能不需要IN蛋白具有酶活性（整合活性）。我们发现IN与逆转录酶之间存在直接的物理相互作用，以及异源HIV-2 IN蛋白无法有效支持逆转录，这表明这种新功能是通过与逆转录起始复合物的其他病毒成分的特异性相互作用而发生的。涉及整合缺陷型和DNA合成缺陷型IN突变体之间互补的研究进一步支持了这一结论，并揭示IN高度保守的HHCC基序对这两种活性都很重要。这些发现为感染细胞内核蛋白逆转录复合物背景下的IN功能和逆转录提供了重要的新见解。此外，它们验证了一种新方法，该方法无需对Gag-Pol进行突变即可在病毒复制水平研究其单个成熟成分的作用。
