Roe T, Chow S A, Brown P O
Department of Biochemistry, Stanford University Medical Center, California 94305, USA.
J Virol. 1997 Feb;71(2):1334-40. doi: 10.1128/JVI.71.2.1334-1340.1997.
Retroviral replication depends on integration of viral DNA into a host cell chromosome. Integration proceeds in three steps: 3'-end processing, the endonucleolytic removal of the two terminal nucleotides from each 3' end of the viral DNA; strand transfer, the joining of the 3' ends of viral DNA to host DNA; and 5'-end joining (or gap repair), the joining of the 5' ends of viral DNA to host DNA. The 5'-end joining step has never been investigated, either for retroviral integration or for any other transposition process. We have developed an assay for 5'-end joining in vivo and have examined the kinetics of 5'-end joining for Moloney murine leukemia virus (MLV). The interval between 3'-end and 5'-end joining is estimated to be less than 1 h. This assay will be a useful tool for examining whether viral or host components mediate 5'-end joining. MLV integrates its DNA only after its host cell has completed mitosis. We show that the extent of 3'-end processing is the same in unsynchronized and aphidicolin-arrested cells. 3'-end processing therefore does not depend on mitosis.
逆转录病毒的复制依赖于病毒DNA整合到宿主细胞染色体中。整合过程分三步进行：3'末端加工，即从病毒DNA的每个3'末端内切核酸酶去除两个末端核苷酸；链转移，即病毒DNA的3'末端与宿主DNA连接；以及5'末端连接（或缺口修复），即病毒DNA的5'末端与宿主DNA连接。5'末端连接步骤从未针对逆转录病毒整合或任何其他转座过程进行过研究。我们开发了一种体内5'末端连接检测方法，并研究了莫洛尼鼠白血病病毒（MLV）5'末端连接的动力学。3'末端和5'末端连接之间的间隔估计小于1小时。该检测方法将是检查病毒或宿主成分是否介导5'末端连接的有用工具。MLV仅在其宿主细胞完成有丝分裂后才整合其DNA。我们表明，在未同步化和阿非迪霉素阻滞的细胞中，3'末端加工的程度是相同的。因此，3'末端加工不依赖于有丝分裂。
