van Gennip René G P, van de Water Sandra G P, Potgieter Christiaan A, van Rijn Piet A
Department of Virology, Central Veterinary Institute of Wageningen University and Research Centre, Lelystad, The Netherlands
Department of Virology, Central Veterinary Institute of Wageningen University and Research Centre, Lelystad, The Netherlands.
J Virol. 2017 Jan 31;91(4). doi: 10.1128/JVI.01328-16. Print 2017 Feb 15.
The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family.
Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.
呼肠孤病毒科由无包膜的多层病毒组成，其双链RNA基因组由9至12个基因组片段组成。呼肠孤病毒科的环状病毒属包含非洲马瘟病毒（AHSV）、蓝舌病毒和流行性出血病病毒，这些病毒会引发应通报的疾病，并通过叮咬库蠓传播。在此，我们利用AHSV的反向遗传学研究由基因组片段2（Seg-2）编码的外衣壳蛋白VP2的作用。Seg-2中先前发现的缺失的扩展表明，AHSV的结构蛋白VP2在体外病毒复制中并非必需。此外，用绿色荧光蛋白（GFP）的RNA序列对Seg-2中的RNA序列进行框内替换，导致AHSV表达GFP，这进一步证实VP2对病毒复制并非必需。与在哺乳动物细胞中不表达VP2的病毒复制相比，在昆虫细胞中的病毒复制显著减少，并且从昆虫细胞中的病毒释放完全被消除。此外，对于不表达VP2的病毒突变体，另一种外衣壳蛋白VP5未与病毒粒子共纯化。然而，不表达VP5的AHSV无法回收，这表明外衣壳蛋白VP5在体外病毒复制中是必需的。我们的结果首次证明，一种病毒结构蛋白在体外对环状病毒复制并非必需，这为呼肠孤病毒科其他成员的研究开辟了新的可能性。
呼肠孤病毒科的成员在全球范围内引发重大健康问题，从人类轮状病毒引起的致命腹泻到不同环状病毒导致的家畜生产经济损失。环状病毒属包含许多病毒种类，其中蓝舌病毒、流行性出血病病毒和非洲马瘟病毒（AHSV）根据世界动物卫生组织的规定会引发应通报的疾病。最近，已表明非结构蛋白NS3/NS3a和NS4在体外病毒复制中并非必需，而通常认为这些无包膜、结构复杂的病毒粒子的结构蛋白VP1至-7是必需的。在此我们首次证明，AHSV的结构蛋白VP2在体外病毒复制中并非必需。我们的发现对于从事无包膜病毒领域研究的病毒学家，特别是呼肠孤病毒学家来说非常重要。
