Understanding Retroviral Reverse Transcription, Recombination, and Replication

在复制过程中，HIV-1将其包装的二聚体RNA基因组转化为DNA，并在感染事件中产生一个原病毒。在这一过程中，HIV-1需要保存其遗传信息，而宿主先天免疫反应试图消除能够产生感染性后代的原病毒的产生。在与Vinay Pathak博士的部门（HIV动力学和复制计划）的合作中，我们研究了由宿主限制性APOBEC3蛋白产生的高突变HIV-1基因组是否可以通过重组或仅遭受亚致死突变来拯救。我们的实验数据和分析表明，这两种情况很少发生；因此，超突变主要导致病毒基因组的死端，这对复制病毒种群的多样性没有贡献。此外，我们还协助我们的合作者完成了几项逆转录病毒复制的研究，包括开发更好的细胞间感染报告，跟踪感染细胞中的HIV-1复合体，表征APOBEC3G和APOBEC3F限制因子，以及研究异嗜性小鼠白血病病毒相关病毒的前体。我们当前和未来的研究工作将集中在检查重组是否是维持HIV-1基因组完整性的关键机制，并确定导致HIV-1重组热点的机制。这些研究试图了解HIV-1如何维持其基因组并重组突变以增加病毒多样性。__背景：HIV-1将两个拷贝的病毒RNA打包成一个病毒粒子，尽管每个RNA包含病毒复制所需的所有信息。长期以来，人们一直推测包装两个病毒RNA拷贝的选择性优势在于从RNA断裂中拯救遗传信息，并允许重组以分类突变并增加病毒多样性。这两种功能虽然经常被假设，但尚未经过实验检验；我们在研究中已经解决了这些假设。当病毒编码的RT使用每个共包装RNA的一部分作为模板时，在DNA合成过程中会发生频繁的逆转录病毒重组。由此产生的DNA在其基因组成中是镶嵌的，并且包含来自每个共包装病毒RNA分子的部分序列。重组可发生在所有HIV-1颗粒中；然而，与亲本不同的后代是由含有两种不同rna的杂合粒子产生的。杂合子颗粒是由被一种以上病毒共感染的细胞和由来自两种不同原病毒的rna共包装而产生的。虽然重组可以发生在纯合子颗粒中，其中包含来自同一原病毒的两个RNA拷贝，但所产生的重组体具有与亲本病毒相同的基因型。已经观察到重组发生在整个HIV-1基因组中；然而，重组热点已被报道并被认为是由RNA结构引起的。我们正在研究RNA结构和重组热点之间的关系。重组在HIV-1 DNA合成过程中经常发生。虽然重组在产生病毒多样性中的作用已被确立，但尚不清楚重组是否需要完成DNA合成。我们通过阻断病毒基因组的一部分重组，研究了重组在病毒复制中的作用。我们发现当重组被阻断时，病毒滴度降低。此外，剩余的原病毒有很高的缺失率。这些研究表明重组是一种挽救病毒遗传信息和维持基因组完整性的机制。据我们所知，这是首次证明重组是HIV-1复制的基本修复机制。这些研究不仅揭示了逆转录病毒采用假二倍体复制策略的好处，也揭示了这些病原体的潜在弱点，这些弱点可能用于开发抗病毒策略。__宿主限制性APOBEC3 （A3）蛋白可以诱导G-to-A超突变。目前，大量的研究对病毒遗传多样性和进化的超突变的贡献存在分歧。在与Pathak小组的合作中，我们确定了高突变对HIV-1重组率的影响，以及通过重组产生活的基因组和促进遗传变异来拯救高突变序列的频率。我们发现，高突变对重组率没有显著影响，在杂合病毒粒子中，高突变与野生型基因组之间的重组仅使病毒突变率增加3.9 x 10-5个突变/bp/复制周期，这与HIV-1的突变率相似。由于高突变和野生型基因组的共包装在体内很少发生，因此高突变和野生型基因组之间的重组对复制HIV-1的遗传变异没有显著影响。我们还分析了先前报道的来自感染患者的超突变序列，并确定A3G和A3F的亚致死突变频率可以忽略不计（1 × 10-11），其对病毒突变的贡献远远低于由逆转录引入的突变。总之，我们得出结论，a3诱导的高突变对HIV-1遗传变异的贡献低于易出错复制过程中突变的贡献。目前，我们正在研究A3是否影响逆转录过程。

During replication, HIV-1 converts its packaged dimeric RNA genomes into DNA and generates one provirus in an infection event. In this process, HIV-1 needs to preserve its genetic information while the host innate immune response attempts to abolish the generation of proviruses capable of producing infectious progeny. In collaboration with Dr. Vinay Pathak's section (HIV Dynamics and Replication Program), we examined whether HIV-1 genomes with hypermutation generated by host restriction APOBEC3 proteins could be rescued by recombination or only suffered sublethal mutations. Our experimental data and analyses demonstrated that both scenarios occurred rarely; therefore, hypermutation mostly results in dead-end viral genomes that do not contribute to the diversity of the replicating viral population. Additionally, we have assisted our collaborators in completing several studies on retroviral replication, including developing better reporters for cell-to-cell infection, following the HIV-1 complex in infected cells, characterizing the restriction factors APOBEC3G and APOBEC3F, and studying the precursors of xenotropic murine leukemia virus-related viruses. Our current and future research efforts are focused on examining whether recombination is a key mechanism to maintain HIV-1 genome integrity and defining the mechanism that causes HIV-1 recombination hot spots. These studies seek to understand how HIV-1 maintains its genome and reassorts mutations to increase viral diversity. __BACKGROUND: HIV-1 packages two copies of viral RNA into one virion, although each RNA contains all of the information required for viral replication. It has long been speculated that the selective advantages for packaging two copies of viral RNA are to rescue the genetic information from breaks in the RNAs and to allow recombination to assort mutations and increase viral diversity. Although often assumed, these two functions have not been tested experimentally; we have addressed these hypotheses in our research. Frequent retroviral recombination occurs during DNA synthesis when virally encoded RT uses a portion of each copackaged RNA as a template. The resulting DNA is mosaic in its genetic composition and contains portions of its sequence from each copackaged viral RNA molecule. Recombination can occur in all HIV-1 particles; however, progeny distinct from parents are generated from heterozygous particles that contain two different RNAs. Heterozygous particles are produced from cells coinfected with more than one virus and by copackaging RNAs derived from two different proviruses. Although recombination can occur in homozygous particles, which contain two copies of RNA derived from the same provirus, the resulting recombinants have the same genotype as the parent virus. Recombination has been observed to occur throughout the HIV-1 genome; however, recombination hot spots have been reported and are thought to be caused by RNA structures. We are examining the relationship between RNA structures and recombination hot spots. __ACCOMPLISHMENTS: Recombination occurs frequently during the synthesis of HIV-1 DNA. Although the role of recombination in generating viral diversity has been established, it is unknown whether recombination is required to complete DNA synthesis. We have investigated the role of recombination in viral replication by blocking recombination in a portion of the viral genome. We found that viral titer decreased when recombination is blocked. Furthermore, the remaining proviruses have high deletion rates. These studies indicate that recombination is a mechanism to salvage viral genetic information and maintain genome integrity. To our knowledge, this is the first demonstration that recombination is an essential repair mechanism for HIV-1 replication. These studies not only reveal the benefit of the pseudodiploid replication strategy employed by retroviruses but also expose the potential weakness of these pathogens, which may be used to develop antiviral strategies. __Host restriction APOBEC3 (A3) proteins can induce G-to-A hypermutation. Currently, numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. In collaboration with the Pathak group, we determined the effects of hypermutation on the HIV-1 recombination rate and how often hypermutated sequences could be rescued via recombination to produce viable genomes and contribute to genetic variation. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 x 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (1 x 10-11) and its contribution to the viral mutations is far below that of mutations introduced by reverse transcription. Overall, we conclude that the contribution of A3-induced hypermutation to HIV-1 genetic variation is lower than the contribution of mutations during error-prone replication. Currently, we are examining whether A3 affects the process of reverse transcription.