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。由于逆转录同时发生在两条链上，并且有可能通过位移合成多次复制同一负链，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。我们将使用先前在我们实验室开发的链特异性定量实时PCR检测来深入了解细胞培养中逆转录的机制。我们将描述NRTI和NNRTI抗性突变对逆转录各步骤动力学的影响。我们将使用链特异性扩增来分析巨噬细胞中的逆转录动力学，以确定较低的dNTP池是否会导致较慢的逆转录。我们将开发一种用于病毒脱衣的细胞培养试验，并确定脱衣的动力学，脱衣的细胞内位置，以及病毒脱衣与逆转录动力学之间的关系。[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 on 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. We will use a strand-specific quantitative real-time PCR assay previously developed in our lab to gain insights into the mechanisms of reverse transcription in cell culture. We will characterize the effects of NRTI and NNRTI resistance mutations on the kinetics of various steps in reverse transcription. We will analyze the kinetics of reverse transcription in macrophages using strand-specific amplification to determine whether lower dNTP pools result in slower reverse transcription. We will develop a cell culture assay for viral uncoating and determine the kinetics of uncoating, the intracellular location of uncoating, and the relationship between viral uncoating and the kinetics of reverse transcription. [Corresponds to Pathak Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]