List J, Haase A T
Department of Microbiology, University of Minnesota, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, USA.
Virology. 1997 Oct 27;237(2):189-97. doi: 10.1006/viro.1997.8785.
Proviral integration is thought to be an obligate step of the retroviral replication cycle but the lentivirus visna has been reported to replicate in sheep choroid plexus (SCP) cultures in the absence of proviral integration. Because of new evidence that visna virus has a functional integrase, we reexamined visna virus infection of SCP cultures and found that proviral integration does indeed occur in this setting. While the majority of viral DNA remains unintegrated, integrated proviruses arise early in infection and accumulate over time. The sequences of the resulting host-virus DNA junctions show that, like other retroviruses, visna loses terminal nucleotides from its DNA upon integration. However, unlike other retroviruses, in over half the host-U3 junctions analyzed only a single nucleotide was lost such that the universally conserved CA dinucleotide, two nucleotides from the end of unintegrated viral DNA, did not directly abut host sequences in the provirus. We analyzed the role of integration in visna replication by introducing a series of five mutations into the integrase gene of molecularly cloned visna virus LV1-1KS1. Each mutation abolished viral replication, suggesting that integration may be an obligatory step in replication. We also documented productive infection of SCP cultures in which cell division had been blocked by g-irradiation. The ability of visna to integrate and to replicate in nondividing cells points to the possible utility of visna-based vectors for gene transfer into differentiated cells.
前病毒整合被认为是逆转录病毒复制周期中的一个必要步骤，但据报道，慢病毒维斯纳病毒（visna virus）在没有前病毒整合的情况下也能在绵羊脉络丛（SCP）培养物中复制。由于有新证据表明维斯纳病毒具有功能性整合酶，我们重新检测了SCP培养物中的维斯纳病毒感染情况，发现在此情况下确实会发生前病毒整合。虽然大多数病毒DNA仍未整合，但整合的前病毒在感染早期出现并随时间积累。由此产生的宿主-病毒DNA连接序列表明，与其他逆转录病毒一样，维斯纳病毒在整合时会从其DNA中丢失末端核苷酸。然而，与其他逆转录病毒不同的是，在分析的超过一半的宿主-U3连接中，仅丢失了一个核苷酸，以至于未整合病毒DNA末端的两个核苷酸处普遍保守的CA二核苷酸在整合后并不直接与前病毒中的宿主序列相邻。我们通过在分子克隆的维斯纳病毒LV1-1KS1的整合酶基因中引入一系列五个突变，分析了整合在维斯纳病毒复制中的作用。每个突变都消除了病毒复制，这表明整合可能是复制中的一个必要步骤。我们还记录了在经γ射线照射使细胞分裂受阻的SCP培养物中的有效感染情况。维斯纳病毒在非分裂细胞中整合和复制的能力表明，基于维斯纳病毒的载体可能有助于将基因转移到分化细胞中。
