Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus
Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus
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DOI：
10.1128/jvi.73.9.7787-7794.1999
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发表时间：
1999-09-01
影响因子：
5.4
通讯作者：
Hofmann, MA
中科院分区：
文献类型：
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作者：
Moser, C;Stettler, P;Hofmann, MA
为了确定猪瘟病毒（CSFV）自主RNA复制的最低要求，基于CSFV Alfort/187的感染性cDNA克隆，构建了结构蛋白和侧翼非结构蛋白基因内具有框内缺失的基因组。在体外从各自的质粒转录RNA并转染到SK-6猪肾细胞中。通过对转染细胞进行CSFV NS 3蛋白免疫染色和通过分析细胞提取物的病毒RNA以及转染后不同时间的蛋白质合成来确定RNA的复制能力。编码N-Pro、C、E-rns、E1、E2、p7和NS 2的基因被证明对于RNA复制是不稳定的，但是复制效率在各个构建体之间变化很大。含有完整NS 2-NS 3基因的RNA复制子在转染细胞中持续存在，并继续复制，而不会对细胞造成任何明显的形态或功能损伤，而缺乏NS 2基因的基因组复制更有效，并诱导细胞病变效应。这些发现表明，尽管NS 2对于瘟病毒RNA复制不是必需的，但它在其中具有调节功能。致细胞病变和非致细胞病变复制子都被包装到由共转染的全长辅助病毒基因组反式提供的病毒颗粒中。
To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N-Pro, C, E-rns, E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.