Human immunodeficiency virus cDNA metabolism: Notable stability of two-long terminal repeat circles
Human immunodeficiency virus cDNA metabolism: Notable stability of two-long terminal repeat circles
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DOI：
10.1128/jvi.76.8.3739-3747.2002
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发表时间：
2002-04-01
影响因子：
5.4
通讯作者：
Bushman, FD
中科院分区：
文献类型：
--
作者：
Butler, SL;Johnson, EP;Bushman, FD
逆转录病毒复制的早期步骤包括将病毒RNA逆转录产生线性双链cDNA拷贝，然后将病毒cDNA整合到宿主细胞的染色体中。一部分病毒cDNA也可能遵循非生产性途径，即形成环状。在一种途径中，线性cDNA的末端通过细胞的非同源DNA末端连接系统连接在一起，形成两端长末端重复序列（2 - LTR）环。有人认为，在人类免疫缺陷病毒（HIV）感染的细胞中，2 - LTR环会迅速降解，因此2 - LTR环的存在可作为患者正在发生新感染的标志物。基于这一观点，在接受高效抗逆转录病毒治疗成功的患者中检测到2 - LTR环，导致有人提出尽管进行了治疗，病毒复制仍持续存在。我们使用荧光监测PCR（Taqman）来定量感染早期HIV cDNA的代谢情况。与先前的研究相反，我们发现在控制混杂变量的实验中，2 - LTR环实际上相当稳定。因此，依赖于2 - LTR环不稳定性的研究有待重新解释。我们还使用定量PCR方法分析了蛋白酶体抑制剂MG132的作用，结果表明，主要包含已完成cDNA的病毒复合物是蛋白酶体介导降解的主要底物。
Early steps of retroviral replication involve reverse transcription of the viral RNA to yield a linear double-stranded cDNA copy and then integration of the viral cDNA into a chromosome of the host cell. A portion of the viral cDNA can also follow nonproductive pathways in which it becomes circularized. In one pathway, the ends of the linear cDNA become joined together by the cellular nonhomologous DNA end-joining system to form two-long terminal repeat (2-LTR) circles. It has been argued that 2-LTR circles are quickly degraded in human immunodeficiency virus (HIV)-infected cells, allowing the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. Following this idea, detection of 2-LTR circles in patients undergoing successful highly active antiretroviral therapy has led to the proposal that viral replication persists despite treatment. We have used fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cDNA early after infection. Contrary to previous work, we find that 2-LTR circles are actually quite stable in experiments where confounding variables are controlled. Thus, studies relying on the lability of 2-LTR circles are open to reinterpretation. We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation.