Laboratory of Retrovirology, The Rockefeller University, New York, New York, United States of America.
Howard Hughes Medical Institute, The Rockefeller University, New York, New York, United States of America.
PLoS Pathog. 2018 Jan 29;14(1):e1006824. doi: 10.1371/journal.ppat.1006824. eCollection 2018 Jan.
The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice site(s). Overall, our data indicate complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication.
HIV-1 的基因组约有 9.5 千碱基对，其中包含控制病毒复制多个方面的 RNA 序列和结构，包括转录、剪接、核输出、翻译、包装和逆转录。尽管如此，化学探测和其他方法表明，HIV-1 基因组可能包含许多具有未知重要性和功能的 RNA 二级结构。为了确定 HIV-1 基因组中是否存在其他未被发现的、对病毒复制很重要的顺式作用 RNA 元件，我们进行了一项全基因组沉默诱变实验。设计并合成了包含约 50 到 200 个同义突变簇的 16 个突变前病毒，这些突变簇几乎覆盖了整个 HIV-1 蛋白编码序列。对这些突变病毒的分析将它们分为三个表型组。第 1 组突变体表现出接近野生型的复制能力，第 2 组突变体表现出复制缺陷和 RNA 剪接异常，第 3 组突变体在没有明显剪接异常的情况下表现出复制缺陷。这三种表型是由沿病毒基因组分布具有明显区域偏倚的突变引起的，这些导致复制缺陷的突变都降低了未剪接 RNA 的水平。我们详细描述了第 2 组突变体的潜在缺陷。第 2 组突变体可以衍生出能够进行病毒复制的第二部位回复突变体，通常包含降低近端剪接位点利用的点突变。导致第 2 组病毒剪接异常的突变体的定位揭示了几个 RNA 序列的存在，这些序列显然抑制了隐式或规范剪接位点的使用。影响剪接的一些序列分布广泛，而另一些序列可以映射到离散的元件上，位于受影响的剪接位点（或其附近）。总体而言，我们的数据表明，HIV-1 基因组的不同区域存在复杂的 RNA 元件，这些元件对 HIV-1 的剪接进行负调控，从而实现平衡剪接和病毒复制。
