The consequences of reconfiguring the ambisense S genome segment of Rift Valley fever virus on viral replication in mammalian and mosquito cells and for genome packaging.
The consequences of reconfiguring the ambisense S genome segment of Rift Valley fever virus on viral replication in mammalian and mosquito cells and for genome packaging.
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DOI：
10.1371/journal.ppat.1003922
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发表时间：
2014-02
期刊：
影响因子：
6.7
通讯作者：
Elliott RM
中科院分区：
文献类型：
--
作者：
Brennan B;Welch SR;Elliott RM
裂谷热病毒（RVFV，布尼亚病毒科）是一种由蚊子传播的牲畜和人类病原体，主要发现于撒哈拉以南非洲和阿拉伯半岛。病毒基因组包含两个负义（L和M片段）和一个双义（S片段）RNA，编码七种蛋白质。S片段编码负义的核衣壳（N）蛋白和正义的非结构（NS）蛋白，尽管NS不能直接从S片段翻译，而是从特定的亚基因组mRNA翻译。使用反向遗传学，我们产生了一种病毒，命名为rMP 12：S-Swap，其中N蛋白从基因组S RNA内的NS基因座表达，NS从N基因座表达。在感染rMP 12：S-Swap NS的细胞中，相对于N，NS的表达水平高于感染亲本rMP 12病毒的细胞。尽管NS是主要的干扰素拮抗剂和毒力的决定因素，但rMP 12：S-Swap在哺乳动物细胞中的生长减弱，并产生小的空斑表型。在感染的哺乳动物细胞中，NS蛋白丰度的增加不会导致宿主细胞蛋白合成或宿主细胞转录的更快抑制。然而，在培养的蚊子细胞中，rMP 12：S-Swap感染导致细胞死亡，而不是如rMP 12所见的持久性的建立。最后，改变S片段的组成导致基因组与反基因组RNA进入rMP 12：S-Swap病毒体的包装比率不同。我们的研究结果突出了RVFV基因组的可塑性，并提供了一个有用的实验工具，以进一步研究分段基因组的包装机制。裂谷热病毒（RVFV）是一种主要在撒哈拉以南非洲发现的蚊媒布尼亚病毒，可以感染家畜和人类。RVFV具有编码七种蛋白质的三重RNA基因组。最小的（S）片段具有不寻常的双义编码策略，其中两个基因（用于核衣壳N和非结构NS蛋白）在基因组RNA上以相反的方向编码，并从特定的亚基因组mRNA翻译。N是病毒的主要结构蛋白，而NSs是病毒的主要毒力因子。为了研究这种编码排列的生物学意义，我们使用反向遗传学来创建重组病毒，其中N和NS编码序列在S片段上交换。与亲本病毒相比，重组病毒在组织培养细胞中生长较差，并且感染导致其死亡，而不是在昆虫细胞中保持持久性。此外，将修饰的S基因组片段包装到新的病毒颗粒中也发生了改变。我们还表明，外源蛋白可以表达到高水平时，克隆的重组病毒中的NS基因的位置。这些研究对疫苗开发和病媒控制策略具有重要意义。
Rift Valley fever virus (RVFV, family Bunyaviridae) is a mosquito-borne pathogen of both livestock and humans, found primarily in Sub-Saharan Africa and the Arabian Peninsula. The viral genome comprises two negative-sense (L and M segments) and one ambisense (S segment) RNAs that encode seven proteins. The S segment encodes the nucleocapsid (N) protein in the negative-sense and a nonstructural (NSs) protein in the positive-sense, though NSs cannot be translated directly from the S segment but rather from a specific subgenomic mRNA. Using reverse genetics we generated a virus, designated rMP12:S-Swap, in which the N protein is expressed from the NSs locus and NSs from the N locus within the genomic S RNA. In cells infected with rMP12:S-Swap NSs is expressed at higher levels with respect to N than in cells infected with the parental rMP12 virus. Despite NSs being the main interferon antagonist and determinant of virulence, growth of rMP12:S-Swap was attenuated in mammalian cells and gave a small plaque phenotype. The increased abundance of the NSs protein did not lead to faster inhibition of host cell protein synthesis or host cell transcription in infected mammalian cells. In cultured mosquito cells, however, infection with rMP12:S-Swap resulted in cell death rather than establishment of persistence as seen with rMP12. Finally, altering the composition of the S segment led to a differential packaging ratio of genomic to antigenomic RNA into rMP12:S-Swap virions. Our results highlight the plasticity of the RVFV genome and provide a useful experimental tool to investigate further the packaging mechanism of the segmented genome. Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus found primarily in sub-Saharan Africa that can infect both domestic animals and humans. RVFV has a tripartite RNA genome that encodes seven proteins. The smallest (S) segment has an unusual ambisense coding strategy whereby two genes (for the nucleocapsid N and nonstructural NSs proteins) are encoded in opposite orientations on the genomic RNA, and are translated from specific subgenomic mRNAs. N is the major structural protein of the virus while NSs is the major virulence factor. To investigate the biological significance of this coding arrangement, we used reverse genetics to create a recombinant virus in which the N and NSs coding sequences were swapped on the S segment. The recombinant virus grew less well in tissue culture cells compared to the parental virus, and rather than maintain persistence in insect cells, infection resulted in their death. In addition, packaging of the modified S genome segment into new virus particles was altered. We also showed that a foreign protein could be expressed to high levels when cloned in place of the NSs gene in the recombinant virus. These studies have implications for vaccine development and vector control strategies.
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DOI：
10.2174/1874357901004020008
发表时间：
2010-04-22
期刊：
The open virology journal
影响因子：
--
作者：
Bouloy M;Weber F
通讯作者：
Weber F
影响因子：
5.4
作者：
Ikegami, T;Won, S;Makino, S
通讯作者：
Makino, S
影响因子：
5.4
作者：
Habjan, Matthias;Pichlmair, Andreas;Weber, Friedemann
通讯作者：
Weber, Friedemann
影响因子：
3.7
作者：
Gerrard, Sonja R.;Bird, Brian H.;Nichol, Stuart T.
通讯作者：
Nichol, Stuart T.
影响因子：
5.4
作者：
Bird, Brian H.;Albarino, Cesar G.;Nichol, Stuart T.
通讯作者：
Nichol, Stuart T.