Kameoka Masanori, Morgan Max, Binette Marc, Russell Rodney S, Rong Liwei, Guo Xiaofeng, Mouland Andrew, Kleiman Lawrence, Liang Chen, Wainberg Mark A
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2.
J Virol. 2002 Apr;76(8):3637-45. doi: 10.1128/jvi.76.8.3637-3645.2002.
Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.
有人提出，人类免疫缺陷病毒1型反式激活因子（Tat）在逆转录调控中发挥作用。我们之前证明，野生型Tat可通过在病毒生命周期后期抑制逆转录酶（RT）反应来增强病毒感染性，以防止过早合成可能有害的病毒DNA产物。在此，我们对无细胞逆转录反应进行了详细分析，以阐明Tat影响这一过程的机制。我们的结果表明，Tat可抑制非特异性DNA延伸，同时适度影响逆转录的特异性起始阶段。此外，Tat具有RNA退火活性，可促进tRNA与病毒RNA结合。这表明Tat与已知直接参与此过程的病毒核衣壳（NC）蛋白之间存在功能同源性。使用一系列突变Tat蛋白进行的实验表明，Tat富含半胱氨酸的结构域和核心结构域负责抑制DNA延伸，而富含半胱氨酸的结构域、核心结构域和碱性结构域以及C末端结构域中的富含谷氨酰胺区域对于tRNA与病毒RNA基因组的结合都很重要。这些结果表明，Tat在RT反应中至少可发挥两种不同作用，即抑制DNA聚合以及将tRNA与病毒RNA结合。我们认为，Tat的第一种活性可能有助于新一轮感染期间病毒基因组逆转录的整体效率以及增强感染性病毒颗粒的产生。我们推测，第二种活性表明Tat与NC之间存在功能同源性，这暗示了NC在病毒成熟过程中取代Tat的潜在作用。
