Herzig Eytan, Voronin Nickolay, Kucherenko Nataly, Hizi Amnon
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
J Virol. 2015 Aug;89(16):8119-29. doi: 10.1128/JVI.00809-15. Epub 2015 May 20.
The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.
Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.
逆转录病毒中的逆转录（RTN）过程对病毒生命周期至关重要。这一关键过程仅由病毒逆转录酶（RT）催化，该酶通过其DNA聚合酶活性将病毒RNA复制为DNA，同时通过其核糖核酸酶H活性去除原始RNA模板。在RTN过程中，DNA合成与RNA水解之间的结合导致链转移（或模板转换），这对RTN的完成至关重要。这些由RT驱动的活动之间的平衡被认为是链转移的唯一原因。然而，我们在此表明，HIV-1 RT中的一个特定突变（L92P）并不影响DNA聚合酶和核糖核酸酶H活性，但却消除了链转移。我们最近发现，RT的链转移完全丧失与RT的DNA钳活性丧失之间也存在良好的相关性。这一发现表明这两种功能之间存在机制联系，并且它们都是RT的直接和独特功能（除了DNA合成和RNA降解）。此外，当将RT的L92P突变体引入感染性HIV-1克隆时，由于RTN期间细胞内链转移效率低下，它失去了病毒复制能力，从而支持了体外数据。据我们所知，这是关于特异性且直接损害与RT相关的链转移的RT突变体的首次报道。因此，靶向亮氨酸92残基可能有助于选择性地阻断这种RT活性，从而抑制HIV-1的感染性和发病机制。
逆转录病毒中的逆转录对于病毒生命周期至关重要。这个多步骤过程由病毒逆转录酶催化，该酶通过其DNA聚合酶活性将病毒RNA复制为DNA（同时通过其核糖核酸酶H活性去除RNA模板）。合成与水解之间的结合和平衡导致链转移，这对逆转录的完成至关重要。我们在此首次表明，HIV-1逆转录酶中的单个突变（L92P）选择性地消除了链转移，而不影响该酶的DNA聚合酶和核糖核酸酶H功能。当将此突变引入感染性HIV-1克隆时，由于细胞内链转移受损，病毒复制丧失，从而支持了体外数据。因此，找到靶向HIV-1逆转录酶亮氨酸92的新型药物可能有助于开发新的强效且选择性的逆转录病毒逆转录抑制剂，从而阻碍HIV-1的感染性。
