Department of Medicine, University of Cambridge, Cambridge, United Kingdom.
J Virol. 2012 May;86(10):5867-76. doi: 10.1128/JVI.00124-12. Epub 2012 Mar 14.
A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5' ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich ((392)-GGAG-(395)) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the (392)-GGAG-(395) motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.
逆转录病毒的一个独特特征是将其基因组的两个拷贝包装在一起，这两个拷贝通过其 5' 端非共价连接。在体外，人免疫缺陷病毒 2 型（HIV-2）RNA 的二聚化通过茎环 1（SL-1）环中暴露的自我互补序列的相互作用发生，该序列也称为二聚化起始位点（DIS）。然而，在病毒粒子中，HIV-2 基因组的二聚化不依赖于 DIS。相反，位于包装信号（Psi）内的回文是基因组二聚化的必需基序。我们之前报道过，降低基因组二聚化和包装效率的 Psi 内突变也会导致成熟颗粒的比例降低（A. L'Hernault、J. S. Greatorex、R. A. Crowther 和 A. M. Lever，Retrovirology 4：90，2007）。在这项研究中，我们通过在 SL-1 中进行一系列靶向突变，进一步研究了 HIV-2 基因组二聚化、颗粒成熟和感染性之间的关系。我们的结果表明，破坏 Psi 内富含嘌呤的((392)-GGAG-(395))基序会严重降低基因组二聚化并导致复制缺陷。保持扩展的 SL-1 结构与（392)-GGAG-(395)基序相结合增强了包装。与 DIS 突变仍能复制的 HIV-1 不同，HIV-2 的复制严重依赖于基因组二聚化，而不仅仅是包装效率。HIV-2 二聚化突变体中的 Gag 加工发生改变，导致 MA-CA-p2 加工中间产物的积累，表明基因组二聚化与颗粒组装之间存在联系。对回复 SL-1 突变病毒的分析表明，基质（70TI）中的补偿性突变可以拯救病毒复制，并部分恢复基因组二聚化和 Gag 加工。我们的结果与 HIV-2 RNA 二聚化和 Gag 多蛋白正确蛋白水解切割之间的相互依赖性一致。
