Molecular Genetics Of Mammalian Retrovirus Replication

逆转录是逆转录病毒（如HIV-1）将其遗传物质（单链RNA）转化为整合到宿主染色体DNA中的双链DNA拷贝的过程。这个过程是复杂的，并由病毒体相关酶，逆转录酶（RT）催化。然而，另一种病毒蛋白，核衣壳蛋白（NC），也需要有效和特异性的病毒DNA合成。 (A)我们研究了NCs活性的机制基础。HIV-1 NC是一种小的碱性核酸结合蛋白，具有两个锌指，每个锌指含有不变的CCHC锌配位基序。它是核酸分子伴侣，即，它具有催化构象重排的能力，所述构象重排导致最稳定的核酸结构。这种性质对于促进合成全长正链和负链病毒DNA所需的两链转移事件至关重要。在负链转移中，逆转录的初始产物（-）强终止DNA在通过存在于RNA和DNA配偶体末端的互补重复区的碱基配对促进的反应中易位到病毒RNA（称为受体RNA）的3 ′末端。(i)使用突变分析，我们现在已经获得的证据表明，NC核酸分子伴侣活性最终取决于退火成核位点处受体RNA局部结构的稳定性，而不是结构的整体稳定性。(ii)我们还表明，NC分子伴侣活性与RNA酶切割一起阻断了正链DNA合成过程中非多嘌呤段RNA的错误引发。这些研究结果表明，NC在确保正链起始的保真度方面具有以前未被认识到的作用。 (B)我们对可能影响HIV-1逆转录的宿主蛋白质的兴趣使我们研究了人APOBEC 3G（A3 G），这是一种具有两个锌指结构域的细胞胞苷脱氨酶，它在不存在病毒蛋白Vif的情况下阻断HIV-1逆转录和复制。抗病毒作用已被证明在很大程度上是脱氨酶依赖性的，但也有一个脱氨酶独立的组件。(i)我们的A3 G研究的一个重点是阐明A3 G抑制逆转录的机制。我们已经成功地纯化了催化活性A3 G，使我们能够提供其脱氨酶和核酸结合活性的全面分子分析。例如，我们已经证明A3 G和NC不干扰彼此与RNA的结合。这表明逆转录的抑制可能与对NC伴侣功能的影响无关。为了验证这一假设，我们研究了A3 G，NC和RT在代表逆转录途径中各个步骤的重构反应中的相互作用。对于第一次，我们已经报道，A3 G不影响NC-介导的退火或RT的RNase H活性的动力学。在鲜明的对比下，A3 G显着抑制所有RT-催化的延伸反应与或不与NC和不需要A3 G催化活性。来自单分子DNA拉伸分析和荧光各向异性的数据支持脱氨酶非依赖性逆转录抑制的新机制，该机制由A3 G、NC和RT的核酸结合性质的关键差异决定。（ii）在目前的工作中，我们已经开始研究APOBEC 3A（A3 A）蛋白，其仅具有一个锌指结构域，并且是通过Line-1和Alu非LTR元件逆转录转座的有效抑制剂。目前正在努力表达和纯化足够大量的蛋白质用于生物化学和结构分析。 (C)我们的实验室也一直在研究HIV-1衣壳蛋白（CA）在早期感染后事件中的作用，这是感染过程中的一个阶段，目前还没有完全了解。(i)我们的初步研究结果阐明了感染性、适当的核心形态、CA蛋白的结构完整性和进行逆转录的能力之间的密切联系。 (ii)最近，我们进行了一项研究，以提供有关CA可塑性的新信息，即，其耐受对CA结构至关重要的疏水残基变化的能力不会完全消除生物活性。突变体的构建和测试，以确定他们是否可能保留复制的能力，从而提出了一个机会，分离第二个网站的抑制。当这些突变体之一W23 F进行长期传代时，分离出第二位点抑制突变W23 F/V26 I，其部分恢复野生型表型。一个结构模型，容纳的空间变化引起的W23 F和V26 I突变可以解释抑制表型。这些发现是新颖的，并表明，尽管CA结构的装配施加的限制，HIV-1是能够部分适应严重的结构扭曲的主要病毒蛋白。(iii)在目前的工作中，我们正在调查的连接器区域，连接CA的N-和C-末端结构域的点突变的影响。我们还研究了两个赖氨酸残基（一个，N-末端，另一个，C-末端）突变的影响，这两个赖氨酸残基被认为对两个CA结构域之间的相互作用很重要。尽管所有突变体都产生病毒颗粒，但大多数在单循环测定中几乎没有或没有感染性。感染性的缺乏与电子显微镜下缺陷核心的出现有关。迄今为止获得的结果表明，一般来说，参与结构域间相互作用的残基不能突变而不丧失适当的结构和功能。对这些突变体的进一步鉴定正在进行中。

Reverse transcription is the process by which a retrovirus such as HIV-1 converts its genetic material (single-stranded RNA) into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for efficient and specific viral DNA synthesis. (A) We study the mechanistic basis for NCs activity. HIV-1 NC is a small, basic nucleic acid binding protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. It is a nucleic acid chaperone, i.e., it has the ability to catalyze conformational rearrangements that lead to the most thermodynamically stable nucleic acid structures. This property is critical for promoting the two strand transfer events that are needed for synthesis of full-length plus- and minus-strand viral DNA. In minus-strand transfer, the initial product of reverse transcription, (-) strong stop DNA, is translocated to the 3-prime end of viral RNA (termed acceptor RNA) in a reaction facilitated by base-pairing of the complementary repeat regions, which are present at the ends of the RNA and DNA partners. (i) Using mutational analysis, we have now obtained evidence demonstrating that NC nucleic acid chaperone activity is ultimately dependent on the stability of acceptor RNA local structure at the nucleation site for annealing, rather than on the overall stability of the structure. (ii) We have also shown that NC chaperone activity together with RNase cleavage block mispriming by non-polypurine tract RNAs during plus-strand DNA synthesis. These findings demonstrate a previously unrecognized role for NC in ensuring the fidelity of plus-strand initiation. (B) Our interest in host proteins that might affect HIV-1 reverse transcription has led us to investigate human APOBEC3G (A3G), a cellular cytidine deaminase with two zinc finger domains, which blocks HIV-1 reverse transcription and replication in the absence of the viral protein known as Vif. The antiviral effect has been shown to be largely deaminase-dependent, but there is also a deaminase-independent component. (i) One focus of our A3G studies has been to elucidate the mechanism for A3G inhibition of reverse transcription. We have succeeded in purifying catalytically active A3G, allowing us to provide a comprehensive molecular analysis of its deaminase and nucleic acid binding activities. We have shown, for example, that A3G and NC do not interfere with each other's binding to RNA. This suggested that inhibition of reverse transcription is likely to be unrelated to an effect on NC chaperone function. To test this hypothesis, we investigated the interplay between A3G, NC, and RT in reconstituted reactions representing individual steps in the reverse transcription pathway. For the first time, we have reported that A3G does not affect the kinetics of NC-mediated annealing or the RNase H activity of RT. In sharp contrast, A3G significantly inhibits all RT-catalyzed elongation reactions with or without NC and without a requirement for A3G catalytic activity. Data from single-molecule DNA stretching analyses and fluorescence anisotropy support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC, and RT. (ii) In current work, we have begun to study the APOBEC3A (A3A) protein, which has only one zinc finger domain and is a potent inhibitor of retrotransposition by Line-1 and Alu non-LTR elements. Efforts are underway to express and purify large enough amounts of protein for biochemical and structural analysis. (C) Our laboratory has also been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. (i) Our initial findings illuminated the intimate connection between infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription. (ii) More recently, we have performed a study to provide new information on the plasticity of CA, i.e., its ability to tolerate changes in hydrophobic residues crucial for CA structure that do not totally abrogate biological activity. Mutants were constructed and tested to determine whether they might retain the ability to replicate and thereby present an opportunity to isolate second-site suppressors. When one of these mutants, W23F, was subjected to long term passage, a second-site suppressor mutation, W23F/V26I was isolated that partially restored the wild-type phenotype. A structural model that accommodates the spatial changes induced by the W23F and V26I mutations can explain the suppressor phenotype. These findings are novel and demonstrate that despite the limits imposed on assembly of CA structure, HIV-1 is able to partially adapt to severe structural distortions in a major viral protein. (iii) In current work, we are investigating the effect of point mutations in the linker region that connects the N- and C-terminal domains of CA. We are also studying the effect of mutations in two lysine residues (one, N-terminal and the other, C-terminal) that are thought to be important for interactions between the two CA domains. Although all of the mutants produce virus particles, most have little or no infectivity in a single-cycle assay. The lack of infectivity is correlated with the appearance of defective cores in the electron microscope. The results obtained thus far indicate that in general, residues involved in interdomain interactions cannot be mutated without loss of proper structure and function. Further characterization of these mutants is in progress.