Wichgers Schreur Paul J, Oreshkova Nadia, Moormann Rob J M, Kortekaas Jeroen
Department of Virology, Central Veterinary Institute, part of Wageningen University and Research Centre, Lelystad, The Netherlands
Department of Virology, Central Veterinary Institute, part of Wageningen University and Research Centre, Lelystad, The Netherlands Department of Infectious Diseases and Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
J Virol. 2014 Sep;88(18):10883-93. doi: 10.1128/JVI.00961-14. Epub 2014 Jul 9.
Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.
Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.
布尼亚病毒基因组由一个小（S）、一个中（M）和一个大（L）的负链RNA片段组成。尽管非编码区已被证明包含转录、复制和衣壳化所需的信号，但驱动至少一个S、M和L片段包装到单个病毒粒子中以产生感染性病毒的机制在很大程度上尚不清楚。裂谷热病毒（RVFV）是布尼亚病毒科最重要的成员之一，可在反刍动物中引起毁灭性疾病，偶尔也会感染人类。我们通过将编码（NSm）GnGc多聚蛋白的糖蛋白前体基因拆分为两个分别编码（NSm）Gn或Gc的单独基因，研究了RVFV基因组包装的灵活性。利用反向遗传学技术，拯救出了六种具有分段糖蛋白前体基因的病毒，从在没有M型片段的情况下包含两个S型片段的病毒到由四个片段组成的病毒（RVFV-4s），其中三个是M型。尽管所有病毒变体都能够在哺乳动物细胞系中生长，但它们无法在蚊子来源的细胞中有效传播。此外，体内研究表明，即使存在主要毒力因子NSs，RVFV-4s也无法在小鼠中引起播散性感染和疾病，但能诱导针对野生型病毒致死性攻击的保护性免疫反应。总之，拆分布尼亚病毒糖蛋白前体基因提供了研究布尼亚病毒基因组包装的新机会，并为开发下一代减毒活布尼亚病毒疫苗提供了新方法。
裂谷热病毒（RVFV）可在反刍动物中引起毁灭性疾病，偶尔也会感染人类。能够产生有效感染的病毒粒子包含小（S）、中（M）和大（L）RNA基因组片段的至少一个拷贝。M片段编码一种糖蛋白前体（GPC）蛋白，该蛋白在共翻译过程中被切割成Gn和Gc，这是病毒进入和融合所必需的。我们研究了RVFV基因组包装的灵活性，并通过应用基于拆分GnGc开放阅读框的独特策略开发了实验性减毒活疫苗。拯救出了几种RVFV变体，从包含两个S型片段的病毒到由四个片段组成的病毒（RVFV-4s），其中三个是M型，并显示它们能诱导快速的保护性免疫反应。总之，布尼亚病毒GPC的分段为研究布尼亚病毒基因组包装提供了一种新方法，并促进了新型减毒活布尼亚病毒疫苗的开发。
