Mechanisms of HIV-1 Reverse Transcription

像所有核酸一样，HIV-1基因组的复制涉及负链和正链的合成。尽管应用了定量真实的时间PCR技术，关于逆转录的复杂性质的几个问题仍然没有答案，包括感染期间RNA和DNA依赖性DNA合成的速率。目前可用的PCR方法无法区分这两条链。由于逆转录同时发生在两条链上，并且具有通过置换合成多次复制相同负链的潜力，因此不可能使用常规PCR分析逆转录的动力学。为了确定感染细胞中HIV-1逆转录的速率，我们开发了一种新的SSA检测方法，该方法使用单链挂锁探针，该探针与负链或正链特异性杂交，连接，并使用实时PCR定量。使用SSA，我们首次确定了293 T和人原代CD 4 + T细胞中HIV-1 RNA依赖性DNA合成的速率。结果显示，293 T细胞和活化的CD 4 + T细胞的负链DNA合成速率基本相同（68 nt/min）。我们还测定了293 T细胞中负链DNA转移（4 min）、正链DNA转移（28 min）和正链DNA合成起始（9 min）的速率。分离3- 4-kb的正链特异性产物以非常相似的动力学积累;因此，不可能测量正链DNA合成的速率。这些结果表明，正链DNA合成是在多个位点启动的。我们还确定了RT抑制剂（AZT，d4 T，ddI和EFV）是否在负链和正链DNA合成过程中表现出不同的作用。结果表明，AZT和d4 T显著抑制RNA依赖的DNA合成，EFV中度抑制，ddI抑制最小。AZT和d4 T在抑制95%病毒复制的浓度下抑制95%负链DNA合成的观察结果表明，AZT和d4 T主要在负链DNA合成期间抑制逆转录。EFV和ddI似乎在正链合成过程中具有更大的抑制作用。这些研究产生了一种新的链特异性PCR技术，应广泛适用于各种分子研究，并提供了在细胞中的HIV-1逆转录过程中的几个关键步骤的第一次测量。[对应于艾滋病毒耐药性方案2007年4月实地访问报告中的Pathak项目3]

Replication of the HIV-1 genome, like all nucleic acids, involves synthesis of a minus and a plus strand. Despite the application of quantitative real time PCR technology, several questions regarding the complex nature of reverse transcription remain unanswered, including the rates of RNA- and DNA-dependent DNA synthesis during infection. Currently available PCR methods cannot distinguish between the two strands. Because reverse transcription occurs of both strands simultaneously, and has the potential to copy the same minus-strand multiple times through displacement synthesis, it is not possible to analyze the kinetics of reverse transcription using conventional PCR. To determine the rates of HIV-1 reverse transcription in infected cells, we have developed a novel SSA assay using single-stranded padlock probes that are specifically hybridized to either the minus strand or the plus strand, ligated, and quantified using real-time PCR. Using SSA, we have determined for the first time the rates of HIV-1 RNA-dependent DNA synthesis in 293T and human primary CD4+ T cells. The results showed that the rates of minus-strand DNA synthesis in 293T cells and in activated CD4+ T cells were essentially identical (68 nt/min). We also determined the rates of minus-strand DNA transfer (4 min), plus-strand DNA transfer (28 min), and initiation of plus-strand DNA synthesis (9 min) in 293T cells. Plus-strand-specific products that were separated by 3- to 4-kb accumulated with very similar kinetics; as a result it was not possible to measure the rate of plus-strand DNA synthesis. These results indicated that plus-strand DNA synthesis is initiated at multiple sites. We also determined whether inhibitors of RT (AZT, d4T, ddI, and EFV) displayed differential effects during minus- and plus-strand DNA synthesis. The results showed that RNA-dependent DNA synthesis was substantially inhibited by AZT and d4T, moderately inhibited by EFV, and minimally inhibited by ddI. The observation that AZT and d4T inhibited minus-strand DNA synthesis by 95% at concentrations that inhibited viral replication by 95% suggests that AZT and d4T inhibit reverse transcription primarily during minus-strand DNA synthesis. EFV and ddI appear to have a greater inhibitory effect during plus-strand synthesis. These studies have generated a novel strand-specific PCR technique that should be widely applicable to a variety of molecular studies, and provide the first measurements of several key steps during HIV-1 reverse transcription in cells. [Corresponds to Pathak Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]