MOLECULAR GENETICS OF MAMMALIAN RETROVIRUS REPLICATION

这个项目的目标是确定所涉及的分子机制 在哺乳动物逆转录病毒的复制中， 了解影响病毒复制早期阶段的因素。 我们一直在使用体外方法来研究反向事件 转录和病毒核衣壳蛋白（NC）在 提高病毒DNA合成的效率和特异性。 我们 已经表明，HIV-1 NC极大地刺激了HIV-1的速率和程度， 负链转移在体外，部分通过抑制形成 由负链强终止DNA产生的自引发产物[（-） SSDNA]。 NC通过使大TAR次级不稳定来发挥这种作用 在（-）SSDNA的3'末端的结构。 的生物学意义 这一先前未被认识到的NC功能得到以下发现的支持， 在使用纯化HIV-1的内源性逆转录酶（RT）试验中 病毒体（含有NC），不能检测到自吸产物。 在 在相关的工作中，我们发现了一种药物， 负链转移在体外和内源性反应。 的 这种药物在抗HIV治疗中的潜在重要性正在被 在HIV-1感染细胞的实验中进行了研究。 其他研究 主要集中在参与启动的核酸-蛋白质相互作用 DNA合成 具有丙氨酸或芳香族氨基酸的RT酶的实验 HIV-1 p66“引物夹”区域残基的取代表明 几乎所有的突变体都在利用RNA+和 负链引物，但能够延长这些DNA版本 引物 我们得出结论，引物夹中的残基经历了独特的 与RNA引物的相互作用，这可能反映了特异性识别 引物中含有RNA的引物-模板的螺旋结构 搁浅。 在使用RNA聚嘌呤束正链的其他研究中， 引物，我们发现，丙氨酸扫描突变的拇指亚结构域 影响RT在引物-模板上的定位并导致RNA酶的损失 H切割特异性。 我们的研究结果还表明，适当的RT定位 不依赖于与引物或模板末端的距离。

The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand factors which influence the early phase of virus replication. We have been using an in vitro approach to study events in reverse transcription and the role of the viral nucleocapsid protein (NC) in increasing the efficiency and specificity of viral DNA synthesis. We have shown that HIV-1 NC greatly stimulates the rate and extent of HIV-1 minus-strand transfer in vitro, in part by suppressing formation of self-priming products generated from minus-strand strong-stop DNA [(-) SSDNA]. NC exerts this effect by destabilizing the large TAR secondary structure at the 3' end of (-) SSDNA. The biological significance of this previously unrecognized NC function is supported by the finding that in endogenous reverse transcriptase (RT) assays with purified HIV-1 virions (which contain NC), self-priming products cannot be detected. In related work, we have found a drug which specifically targets minus-strand transfer in both in vitro and endogenous reactions. The potential importance of this drug for anti-HIV therapy is being investigated in experiments with HIV-1-infected cells. Other studies are focused on nucleic acid-protein interactions involved in initiation of DNA synthesis. Experiments with RT enzymes having alanine or aromatic substitutions in residues of the HIV-1 p66 "primer grip" region show that almost all of the mutants are defective in utilization of RNA plus- and minus-strand primers, but are able to extend DNA versions of these primers. We conclude that residues in the primer grip undergo unique interactions with the RNA primers, which may reflect specific recognition of the helical structure of a primer-template having RNA in the primer strand. In additional studies with the RNA polypurine tract plus-strand primer, we find that alanine-scanning mutations in the thumb subdomain affect RT positioning on the primer-template and lead to a loss of RNase H cleavage specificity. Our results also show that proper RT positioning does not depend on the distance from the ends of the primer or template.