Mechanisms of HIV-1 Reverse Transcription

HIV-1基因组的复制，像所有的核酸一样，包括负链和正链的合成。尽管应用了实时定量PCR技术，但有关逆转录复杂性的几个问题仍未得到解答，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。由于两条链同时发生逆转录，并且有可能通过位移合成多次复制相同的负链，因此不可能使用传统的PCR来分析逆转录的动力学。为了确定HIV-1在感染细胞中的逆转录率，我们开发了一种新的SSA检测方法，使用单链挂锁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293T和人类原代CD4+ T细胞中HIV-1 rna依赖性DNA合成的速率。结果表明，293T细胞和活化CD4+ T细胞的负链DNA合成速率基本相同（68 nt/min）。我们还测定了293T细胞中负链DNA转移（4分钟）、正链DNA转移（28分钟）和正链DNA合成起始（9分钟）的速率。正链特异性产物分离3- 4kb，积累非常相似的动力学；因此不可能测量正链DNA的合成速率。这些结果表明，正链DNA的合成是在多个位点开始的。我们还确定了RT抑制剂（AZT、d4T、ddI和EFV）在负链和正链DNA合成过程中是否表现出不同的作用。结果表明，AZT和d4T能显著抑制rna依赖性DNA合成，EFV能适度抑制rna依赖性DNA合成，ddI能轻微抑制rna依赖性DNA合成。AZT和d4T在抑制病毒复制95%的浓度下抑制负链DNA合成95%，这表明AZT和d4T主要在负链DNA合成过程中抑制逆转录。EFV和ddI在正链合成过程中似乎有更大的抑制作用。这些研究产生了一种新的链特异性PCR技术，该技术应该广泛适用于各种分子研究，并提供了HIV-1细胞逆转录过程中几个关键步骤的首次测量。[对应于2007年4月HIV耐药计划实地考察报告中的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]