Rescue of the prototypic Arenavirus LCMV entirely from plasmid
Rescue of the prototypic Arenavirus LCMV entirely from plasmid
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DOI：
10.1016/j.virol.2006.01.012
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发表时间：
2006-07-05
期刊：
影响因子：
3.7
通讯作者：
de la Torre, Juan C.
中科院分区：
文献类型：
--
作者：
Sanchez, Ana B.;de la Torre, Juan C.
我们记录了一个不依赖于辅助基因的反向遗传学系统，用于从克隆的cDNA中拯救感染性沙粒病毒。我们构建了含有原型沙粒病毒淋巴细胞性脉络丛脑膜炎病毒（LCMV）Armstrong（ARM）株的反基因组（ag）L和S片段的全长cDNA的质粒，其5 '-和3'-末端分别由T7 RNA聚合酶（T7RP）启动子和核酶序列侧接。这些质粒在表达质粒提供的T7Rp的细胞中指导病毒L和Sag RNA种类的细胞内合成。共表达质粒提供的LCMV反式作用因子，核蛋白（NP）和聚合酶（L），导致L和S ag和基因组RNA种类的复制和表达，并产生称为rT7/LCMV的LCMV感染后代。基于在重组S片段中引入的遗传标签，明确鉴定了重组rT7/LCMV。此外，rT7/LCMV表现出ARM样LCMV预测的生长和生物学特性。据我们所知，这是第一个记录的沙粒病毒拯救，以及双义负链（NS）RNA病毒，完全来自克隆的cDNA。我们的研究结果将反向遗传方法用于DNA介导的病毒拯救扩展到所有已知的具有NS RNA基因组的病毒家族。(c)2006爱思唯尔公司All rights reserved.
We document a helper-independent reverse genetics system for rescuing infectious arenaviruses from cloned cDNAs. We constructed plasmids containing full-length cDNAs of the antigenomic (ag) L and S segments of the Armstrong (ARM) strain of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) flanked at their 5 '- and 3 '-termini by the T7 RNA polymerase (T7RP) promoter and ribozyme sequences, respectively. These plasmids directed intracellular synthesis of viral L and S ag RNA species in cells expressing plasmid-supplied T7Rp. Co-expression of plasmid-supplied LCMV trans-acting factors, nucleoprotein (NP) and polymerase (L), resulted in replication and expression of L and S ag and genome RNA species, and generation of LCMV infectious progeny termed rT7/LCMV. The recombinant rT7/LCMV was unequivocally identified based on a genetic tag introduced in the recombinant S segment. In addition, rT7/LCMV exhibited growth and biological properties predicted for an ARM-like LCMV. To our knowledge, this is the first documented Arenavirus rescue, as well as of an ambisense negative strand (NS) RNA virus, entirely from cloned cDNAs. Our results extend the use of reverse genetic approaches for DNA-mediated virus rescue to all known virus families with NS RNA genome. (c) 2006 Elsevier Inc. All rights reserved.