Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York, USA.
Retrovirology. 2010 Sep 8;7:73. doi: 10.1186/1742-4690-7-73.
The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1) sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants.
HIV-1 lacking SL1 (NLΔSL1) did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K) on day 18 postinfection and C1907T in the SP1 domain (P10L) on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1.
HIV-1 evolved to overcome a deletion in SL1 and restored infectivity by acquiring compensatory mutations in the N-terminal matrix or SP1 domain of Gag. These data shed light on the functions of the N-terminal matrix and SP1 domains and suggest that both regions may have a role in Gag interactions with spliced viral RNA.
HIV-1 的病毒基因组包含几个对调节病毒复制很重要的二级结构。5'非翻译区中的茎环 1（SL1）序列指导 HIV-1 基因组 RNA 二聚化并包装到病毒粒子中。没有 SL1，HIV-1 就不能在人 T 细胞系中复制。SL1 缺失突变体的复制限制表型似乎是多因素的，在产生细胞中的病毒 RNA 二聚化和包装以及感染细胞中病毒 RNA 的逆转录中都存在缺陷。在这项研究中，我们试图描述 SL1 突变体的复制限制，并深入了解回复突变体中补偿的潜在机制。
直到第 18 天出现两个独立的非同义突变（基质域中的 G913A（E42K）和 SP1 域中的 C1907T（P10L）），缺乏 SL1 的 HIV-1（NLΔSL1）才在 PM-1 细胞中复制。携带补偿性突变的 NLΔSL1 回复突变体在 PM-1 细胞中显示出增强的感染性。SL1 回复突变体产生的每纳克 p24 的感染性颗粒比 NLΔSL1 多得多。与野生型相比，SL1 缺失突变体在病毒粒子中包装的 HIV-1 基因组 RNA 较少，而细胞 RNA 较多，特别是信号识别粒子 RNA。NLΔSL1 还将 3 到 4 倍的剪接 HIV mRNA 包装到病毒粒子中，可能会干扰感染性病毒的产生。相比之下，两个回复突变体包装的这些 HIV-1 mRNA 种类少 2.5 到 5 倍。用甲醛固定细胞中的 Gag 交联的 RNA 的定量 RT-PCR 分析表明，补偿性突变降低了 Gag 与剪接 HIV-1 RNA 的结合，从而有效地阻止这些 RNA 被包装到病毒粒子中。病毒粒子中剪接病毒 RNA 的减少可能在促进感染性病毒的产生方面起主要作用，从而恢复 NLΔSL1 的感染性。
HIV-1 进化以克服 SL1 缺失，并通过在 Gag 的 N 端基质或 SP1 域中获得补偿性突变来恢复感染性。这些数据揭示了 N 端基质和 SP1 结构域的功能，并表明这两个区域都可能在 Gag 与剪接病毒 RNA 的相互作用中发挥作用。
