van Knippenberg Ingeborg, Elliott Richard M
MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.
MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom
J Virol. 2015 May;89(10):5525-35. doi: 10.1128/JVI.03595-14. Epub 2015 Mar 4.
The Bunyamwera (BUNV) orthobunyavirus NSs protein has proven a challenge to study in the context of viral infection. NSs is encoded in a reading frame that overlaps that of the viral nucleocapsid (N) protein thus limiting options for mutagenesis. In addition, NSs is poorly immunogenic, and antibodies only work in certain techniques while the protein itself is subject to proteasomal degradation. In order to generate a virus that expresses NSs independently of N, an ambisense S RNA segment was designed by mutating the 5'- and 3'-terminal nucleotide sequences. These mutations were previously shown to alter promoter activity so that both replication and transcription were promoted from both the genome and the antigenome RNAs (J. N. Barr et al., J. Virol. 79: 12602-12607, 2005). As proof of principle, a recombinant BUNV was created that expressed green fluorescent protein (GFP) in the ambisense orientation. GFP expression was detected throughout at least 10 passages. Recombinant BUNV encoding epitope-tagged versions of NSs in the ambisense orientation expressed NSs via a subgenomic mRNA, and two viruses grew to titers only modestly lower than parental rBUNdelNSs2 virus. The ambisense viruses were temperature sensitive, and NSs was shown to localize to both the nucleus and the cytoplasm during infection. These viruses will be useful in further studies on structure-function relationships of the orthobunyavirus NSs protein.
Bunyamwera virus (BUNV) is the type species and model system for both the family Bunyaviridae and the genus Orthobunyavirus, a group that includes many significant human and animal pathogens. Studying the basic molecular biology of these viruses is of great importance to underpin research into vaccines and antivirals. We demonstrate here the plasticity of the BUNV genome by generating recombinant viruses where the normal negative-sense S segment has been converted into an ambisense segment, allowing independent expression of either a foreign gene (green fluorescent protein) or the viral nonstructural NSs protein. These new reagents will allow detailed investigation of NSs, the orthobunyavirus interferon antagonist.
已证明布尼亚姆韦拉病毒（BUNV）正布尼亚病毒属的NSs蛋白在病毒感染的背景下进行研究具有挑战性。NSs由一个与病毒核衣壳（N）蛋白的阅读框重叠的阅读框编码，因此限制了诱变的选择。此外，NSs的免疫原性较差，抗体仅在某些技术中起作用，而该蛋白本身会被蛋白酶体降解。为了产生一种独立于N表达NSs的病毒，通过突变5'和3'末端核苷酸序列设计了一个双义S RNA片段。先前已证明这些突变会改变启动子活性，从而促进基因组和反基因组RNA的复制和转录（J.N. Barr等人，《病毒学杂志》79：12602 - 12607，2005年）。作为原理验证，创建了一种重组BUNV，其以双义方向表达绿色荧光蛋白（GFP）。在至少10代中都检测到了GFP表达。以双义方向编码带有表位标签的NSs版本的重组BUNV通过亚基因组mRNA表达NSs，并且两种病毒的生长滴度仅略低于亲本rBUNdelNSs2病毒。双义病毒对温度敏感，并且已证明NSs在感染期间定位于细胞核和细胞质。这些病毒将有助于进一步研究正布尼亚病毒属NSs蛋白的结构 - 功能关系。
布尼亚姆韦拉病毒（BUNV）是布尼亚病毒科和正布尼亚病毒属的模式种和模式系统，该属包括许多重要的人类和动物病原体。研究这些病毒的基础分子生物学对于支持疫苗和抗病毒药物的研究非常重要。我们在此通过产生重组病毒证明了BUNV基因组的可塑性，其中正常的负义S片段已转化为双义片段，从而允许独立表达外源基因（绿色荧光蛋白）或病毒非结构NSs蛋白。这些新试剂将允许对正布尼亚病毒属的干扰素拮抗剂NSs进行详细研究。
