Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo
Recombinant respiratory syncytial viruses with deletions in the NS1, NS2, SH, and M2-2 genes are attenuated in vitro and in vivo
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DOI：
10.1006/viro.2000.0393
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发表时间：
2000-07-20
期刊：
影响因子：
3.7
通讯作者：
Nguyen, N
中科院分区：
文献类型：
--
作者：
Jin, H;Zhou, H;Nguyen, N
呼吸道合胞病毒（RSV）编码几种缺乏明确功能的蛋白质；包括NS1、NS2、SH和M2-2。先前的研究表明，NS2、SH和M2-2都可以从RSV基因组中删除，因此被认为是辅助蛋白。为了确定当两个或多个转录单位被删除时RSV是否能有效复制，我们从人类RSV A2株的感染性cDNA克隆中分别和不同组合地去除NS1、NS2、SH和M2-2基因。获得了6个缺失两个或两个以上基因的突变体：Delta NS1NS2、Delta M2-2SH、Delta M2-2NS2、Delta SHNS1、Delta SHNS2和Delta SHNS1NS2。M2-2和NS1的缺失对RSV的复制是有害的。当所有四个基因都被删除时，不可能获得重组RSV。所有的双重和三重缺失突变体在体外都表现出复制减少和小斑块形态。这些缺失突变体在HEp-2细胞中的复制比在Vero细胞中更少。在获得的10个单基因和多基因缺失突变体中，Delta M2-2NS2减毒最多，Delta M2-2NS2在HEp-2细胞中形成几乎不可见的斑块，与感染HEp-2细胞的野生型重组RSV相比，其滴度降低了3 log（10）。经鼻内接种棉大鼠后，所有缺失突变体在呼吸道中均被减弱。我们的数据表明，NS1、NS2、SH和M2-2蛋白虽然在体外病毒复制中是不可缺少的，但对有效的RSV复制提供了辅助功能，(C) 2000学术出版社。
Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: Delta NS1NS2, Delta M2-2SH, Delta M2-2NS2, Delta SHNS1, Delta SHNS2, and Delta SHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, Delta M2-2NS2 was most attenuated, Delta M2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log(10) compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication, (C) 2000 Academic Press.