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副本的过程。这个过程是复杂的，由病毒粒子相关酶，逆转录酶（RT）催化。然而，另一种病毒蛋白，核衣壳蛋白（NC），也需要确保高效和特异性的病毒DNA合成。我们研究了NC活性的机理基础。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。它是一种核酸伴侣，也就是说，它具有催化构象重排的能力，从而导致最热力学稳定的核酸结构。这种特性对于促进合成全长正链和负链病毒DNA所需的两条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RNA和DNA伴侣端存在的互补重复区域的碱基配对促进的反应中被易位到病毒RNA的3 '端（称为受体RNA）。(A)在最近的NC工作中，我们关注的是核酸结构和热稳定性变化对NC介导的负链转移的影响。我们发现，只有当（-）强阻DNA和受体RNA结构适中，并且这两种反应物之间保持微妙的热力学平衡时，链转移才有效。这一发现与NC是二级结构弱失稳剂的事实是一致的。利用突变分析，我们现在已经获得证据表明，NC核酸伴侣活性最终取决于在成核部位退火的受体RNA局部结构的稳定性，而不是结构的整体稳定性。此外，我们有一个新的发现表明，当需要nc促进受体RNA的不稳定时，退火似乎比链转移（即退火加延伸）更有效。我们通过显示Mg2+来解释这种明显的差异，Mg2+不存在于单独退火的分析中，但必须加入rt催化的延伸，成功地与NC竞争结合到带负电的核酸磷酸二酯主链上。这些研究强调了研究NC伴侣活性的最佳离子条件的重要性。(B)我们最近启动了一个关于人类APOBEC3G （A3G）的新项目，这是一种具有两个锌指结构域的细胞胞苷脱氨酶，在缺乏病毒蛋白Vif的情况下阻断HIV-1的逆转录和复制。抗病毒作用已被证明主要依赖于脱氨酶，但也有脱氨酶不依赖的成分。我们研究A3G的一个重点是阐明A3G抑制逆转录的机制。我们已经成功纯化了具有催化活性的A3G，使我们能够对其脱氨酶和核酸结合活性进行全面的分子分析。我们已经证明A3G只脱氨单链DNA中的胞嘧啶，而它有效地结合单链DNA和RNA。此外，我们还表明A3G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伴侣蛋白功能的影响无关。为了验证这一假设，我们研究了A3G、NC和RT在代表逆转录途径单个步骤的重组反应中的相互作用。我们首次报道了A3G不影响NC介导的退火动力学或rt的RNase H活性。与之形成鲜明对比的是，A3G在不需要A3G催化活性的情况下，在有或没有NC的情况下，显著抑制所有rt催化的延伸反应。涉及单分子DNA拉伸分析和荧光各向异性的合作研究已经进行。这些数据支持了脱氨酶非依赖性反转录抑制的新机制，该机制是由A3G、NC和rt的核酸结合特性的关键差异决定的。(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，这是感染过程中一个尚未完全了解的阶段。最初的研究集中在HIV-1 CA保守的n端疏水残基中与单丙氨酸取代突变相关的不寻常表型上。我们的研究结果说明了CA蛋白的传染性、适当的核心形态、结构完整性和逆转录能力之间的密切联系。(i)最近，我们进行了一项研究，提供了关于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，这些变化不会完全取消其生物活性。我们的方法是制造可能保留复制能力的突变结构，从而提供分离第二位点抑制子的机会。在总共13个单次替换中，只有一个突变体W23F在单周期试验中显示出传染性，尽管水平很低。W23F能够在MT-4细胞的长期培养中复制，但复制动力学延迟。通过继续传代，我们最终可以分离出具有第二位点抑制突变W23F/V26I的病毒粒子，这部分恢复了野生型表型。一个能够适应W23F和V26I突变引起的空间变化的结构模型表明，Phe23和Ile26之间可能存在疏水相互作用，并且可以解释抑制表型。（iii）在目前的工作中，我们正在研究连接CA的N端和c端结构域的连接子区域点突变的影响。我们也对两个赖氨酸残基（一个N端和另一个c端）突变的影响感兴趣，在交联研究的基础上，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。到目前为止，突变表型似乎没有由保守疏水残基变化引起的突变表型严重。

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 to ensure efficient and specific viral DNA synthesis. We study the mechanistic basis for NC 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. (A) In recent work on NC, we have focused on the effect of changes in nucleic acid structure and thermostability on NC-mediated minus-strand transfer. We found that strand transfer is efficient only when (-) strong-stop DNA and acceptor RNA are moderately structured and a delicate thermodynamic balance between these two reactants is maintained. This finding is consistent with the fact that NC is a weak destabilizer of secondary structure. 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. In addition, we have made a novel finding showing that when NC-facilitated destabilization of acceptor RNA is required, annealing appears to be more efficient than strand transfer (i.e., annealing plus elongation). We explain this apparent discrepancy by showing that Mg2+, which is not present in assays for annealing alone, but must be added for RT-catalyzed elongation, successfully competes with NC for binding to the negatively charged phosphodiester backbone of the nucleic acids. These studies underscore the importance of having optimal ionic conditions for studies of NC chaperone activity. (B) We have recently initiated a new project on 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. 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 demonstrated that A3G deaminates cytosines in single-stranded DNA only, whereas it binds efficiently to single-stranded DNA and RNA. Moreover, we have also shown that A3G and NC do not interfere with each others 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 report 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, without a requirement for A3Gs catalytic activity. Collaborative studies involving single-molecule DNA stretching analyses and fluorescence anisotropy have been performed. The data 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. (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. Initial efforts focused on the unusual phenotype associated with single alanine substitution mutations in conserved N-terminal hydrophobic residues of HIV-1 CA. Our findings illustrated the intimate connection between infectivity, proper core morphology, structural integrity of the CA protein, and the ability to undergo reverse transcription. (i) 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. Our approach was to make mutant constructs that might retain the ability to replicate and thereby present an opportunity to isolate second-site suppressors. Of a total of 13 single substitutions, only one mutant, W23F, was found to exhibit infectivity in a single-cycle assay, albeit at a very low level. W23F was able to replicate during long-term culture in MT-4 cells, but with delayed replication kinetics. With continued passage, we could eventually isolate virions with a second-site suppressor mutation, W23F/V26I, which partially restored the wild-type phenotype. A structural model that accommodates the spatial changes induced by the W23F and V26I mutations shows that hydrophobic interactions between Phe23 and Ile26 are possible and can explain the suppressor phenotype. (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 interested in the effect of mutations in two lysine residues (one, N-terminal and the other, C-terminal) that on the basis of cross-linking studies are thought to be important for interactions between the two CA domains. Thus far, the mutant phenotypes appear to be less severe than those resulting from changes in the conserved hydrophobic residues.