Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs
Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs
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DOI：
10.1128/jvi.79.16.10420-10428.2005
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发表时间：
2005-08-01
影响因子：
5.4
通讯作者：
Weber, F
中科院分区：
文献类型：
--
作者：
Blakqori, G;Weber, F
拉克罗斯病毒（LACV）属于布尼亚病毒科，可引起儿童严重脑炎。它有一个负义RNA基因组，由三个片段L，M和S组成。我们使用先前为Bunyamwera病毒建立的系统，通过仅转染三种质粒成功地拯救了LACV（Lowen et al.，Virology 330：493-500，2004）。这些cDNA质粒代表反基因组方向的三个病毒RNA片段，由T7 RNA聚合酶在细胞内转录，3'端由丁型肝炎病毒核酶修剪。正如布尼亚姆韦拉病毒所显示的，反基因组质粒既可以作为反基因组RNA的供体，也可以作为支持质粒，为RNA固定和颗粒形成提供少量的病毒蛋白。然而，与其他拯救系统相反，转染额外的支持质粒完全废除了拯救，表明LACV对病毒蛋白的过表达高度敏感。组成型表达T7 RNA聚合酶的BSR-T7/5细胞系允许LACV的有效拯救，每毫升产生约10（8）个感染性病毒。通过产生含有遗传标记的野生型病毒（rLACV）和在S片段上具有缺失的NS基因的突变体（rLACVdelNS）来证明该系统的实用性。表达NS的rLACV形成清晰的斑块，显示出有效的宿主细胞关闭，并强烈促凋亡。相比之下，rLACVdeINSs突变体表现出混浊斑块表型和不太明显的关闭，并诱导很少的细胞凋亡。然而，两种病毒在Vero细胞中生长至相似滴度。我们的反向遗传学系统现在使我们能够操纵LACV的基因组，以表征其毒力因子并开发潜在的候选疫苗。
La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3' ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 10(8) infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdeINSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.