Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids
Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids
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DOI：
10.1099/vir.0.18876-0
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发表时间：
2003-05-01
影响因子：
3.8
通讯作者：
Weber, F
中科院分区：
文献类型：
--
作者：
Blakqori, G;Kochs, G;Weber, F
拉克罗斯病毒 (LACV) 属于布尼亚病毒科，是儿科疾病的主要原因；美国脑炎。本研究克隆了LACV的功能性RNA聚合酶Q基因，并建立了反向遗传学系统。通过将基因组 M 片段的 3' 和 5' 非编码区置于海肾荧光素酶基因的侧翼，构建了模仿病毒基因组的报告微型复制子。这些非编码区充当病毒聚合酶的启动子。 L 和核衣壳 (N) 基因均通过 T7 RNA 聚合酶表达，该酶由表达重组 T7 的修饰痘苗病毒安卡拉提供。海肾报告基因在转染细胞中的活性反映了功能性 L 和 N 基因产物的重组核衣壳的重建。时程实验表明，L 和 N 表达开始后 10 至 18 小时，微型复制子活性迅速增加。发现微型复制子活性取决于 L 与 N 质粒的正确比例，任一构建体过多都会导致下调。此外，还发现了 LACV NSS 蛋白对微型复制子活性的特异性抑制作用。在使用亲本辅助病毒粒子的传代实验中，证明重组核衣壳是 LACV 转录、复制和包装的有用模型。
La Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric; encephalitis in the United States. In this study, a functional RNA polymerase Q gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3' and 5' noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSS protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.