van Gennip René G P, van de Water Sandra G P, van Rijn Piet A
Central Veterinary Institute of Wageningen UR (CVI), Department of Virology, Lelystad, The Netherlands.
PLoS One. 2014 Jan 20;9(1):e85788. doi: 10.1371/journal.pone.0085788. eCollection 2014.
Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG(Met)→GCC(Ala) mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.
环状病毒是呼肠孤病毒科中最大的属，由至少23种不同的病毒种类组成。其中之一是蓝舌病毒（BTV），可在反刍动物中引起严重的出血性疾病，并通过库蠓叮咬传播。BTV是一种无包膜病毒，通过细胞裂解和/或由基因组片段10（Seg-10）编码的非结构蛋白NS3/NS3a诱导的独特出芽过程从受感染细胞中释放出来。NS3和NS3a在由库蠓传播的环状病毒中高度保守，这表明它们在病毒复制中起着至关重要的作用。我们利用反向遗传学技术构建了BTV突变体，以研究NS3/NS3a在病毒复制中的功能。最初，Seg-10中带有小插入片段的BTV没有出现细胞病变效应（CPE），但经过几代传代后，这些BTV突变体恢复为与野生型BTV相当的CPE表型，并且通过开放阅读框（ORF）的修复恢复了NS3/NS3a的表达。这些结果表明，功能性NS3/NS3a存在强烈的选择。为了消除NS3和/或NS3a的表达，使用带有一个或两个突变起始密码子（mutAUG1、mutAUG2和mutAUG1+2）的Seg-10构建了BTV突变体。令人惊讶的是，所有三种BTV突变体都成功构建，并且各自的AUG（Met）→GCC（Ala）突变得以维持。通过蛋白质免疫印迹分析以及用NS3/NS3a单克隆抗体对感染细胞进行免疫染色，证实了NS3、NS3a或两者蛋白的表达缺失。mutAUG1和mutAUG1+2病毒在BSR细胞中的生长在昆虫细胞和哺乳动物细胞中均受到抑制，特别是从昆虫细胞中释放的病毒量大幅减少。我们的研究结果现在能够对Seg-10的RNA序列独立于已知基因产物的作用以及NS3/NS3a蛋白在两种类型细胞以及宿主和昆虫载体中的功能进行研究。
