Understanding Retroviral Reverse Transcription, Recombination, and Replication

在复制过程中，HIV-1将其包装的二聚体RNA基因组转换为DNA，并在感染事件中生成一个前病毒。在这个过程中，HIV-1需要保存自己的遗传信息，而宿主的先天免疫反应试图消除能够产生传染性后代的前病毒的产生。我们与Vinay Pathak博士的研究室(HIV动力学和复制计划)合作，研究了宿主限制APOBEC3蛋白产生的高度突变的HIV-1基因组是否可以通过重组拯救，或者只发生亚致死突变。我们的实验数据和分析表明，这两种情况很少发生；因此，超突变主要导致死胡同的病毒基因组，而这些基因组不利于复制的病毒种群的多样性。此外，我们还协助我们的合作者完成了几项关于逆转录病毒复制的研究，包括开发更好的细胞间感染报告程序，跟踪感染细胞中的HIV-1复合体，表征限制因子APOBEC3G和APOBEC3F，以及研究异嗜性小鼠白血病病毒相关病毒的前体。我们目前和未来的研究重点是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重组的机制热点。这些研究试图了解HIV-1如何维持其基因组并对突变进行重新排序，以增加病毒多样性。__背景：HIV-1将两个拷贝的病毒RNA包装成一个病毒粒子，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，包装两个拷贝的病毒RNA的选择性优势是将遗传信息从RNA的断裂中拯救出来，并允许重组以分类突变和增加病毒多样性。虽然经常假设，但这两个功能还没有经过实验测试；我们在研究中解决了这些假设。在DNA合成过程中，当病毒编码的RT使用每个共包装的RNA的一部分作为模板时，频繁的逆转录病毒重组发生。由此产生的DNA在其基因组成上是镶嵌的，并包含来自每个套装病毒RNA分子的部分DNA序列。重组可能发生在所有HIV-1颗粒中；然而，与父母不同的后代是由包含两个不同RNA的杂合颗粒产生的。杂合颗粒是由与一种以上病毒共感染的细胞和来自两种不同前病毒的共同包装的RNA产生的。虽然重组可以发生在纯合子颗粒中，其中包含来自同一前病毒的两个副本的RNA，但得到的重组体具有与母病毒相同的基因。已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。__成就：在HIV-1DNA的合成过程中，重组频繁发生。虽然重组在产生病毒多样性中的作用已经确定，但是否需要重组才能完成DNA合成尚不清楚。我们通过阻断部分病毒基因组中的重组，研究了重组在病毒复制中的作用。我们发现，当重组被阻止时，病毒滴度会下降。此外，剩余的前病毒具有很高的缺失率。这些研究表明，重组是挽救病毒遗传信息和保持基因组完整性的一种机制。据我们所知，这是第一次证明重组是HIV-1复制的必要修复机制。这些研究不仅揭示了逆转录病毒采用的伪二倍体复制策略的好处，而且也暴露了这些病原体的潜在弱点，这可能被用来开发抗病毒策略。__宿主限制性APOBEC3(A3)蛋白可诱导G-to-A超突变。目前，关于超突变对病毒遗传多样性和进化的贡献，许多研究都存在分歧。在与Pathak小组的合作中，我们确定了超突变对HIV-1重组率的影响，以及通过重组来挽救高突变序列以产生可存活的基因组并促成遗传变异的频率。我们发现，高突变对病毒的重组率没有显著影响，在杂合病毒粒子中，高突变和野生型基因组之间的重组仅使病毒变异率增加3.9×10-5个突变/个碱基/复制周期，这与HIV-1的变异率相似。由于高突变和野生型基因组在体内很少发生共包装，因此高突变和野生型基因组之间的重组对复制的HIV-1的遗传变异没有显著的贡献。我们还分析了以前报道的感染患者的高突变序列，确定A3G和A3F的亚致死性突变频率可以忽略不计(1×10-11)，它对病毒突变的贡献远远低于反转录引入的突变。总体而言，我们得出的结论是，A3诱导的超突变对HIV-1基因变异的贡献低于易于错误复制的突变的贡献。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.