Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, Scotland, UK.
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh EH25 9RG, Scotland, UK.
J Gen Virol. 2012 Apr;93(Pt 4):840-849. doi: 10.1099/vir.0.039966-0. Epub 2011 Dec 21.
The influenza A virus genome comprises eight segments of negative-sense RNA that encode up to 12 proteins. RNA segment 2 encodes three proteins, PB1, PB1-F2 and N40, that are translated from the same mRNA by ribosomal leaky scanning and reinitiation. PB1 is a subunit of the trimeric viral RNA polymerase. PB1-F2 has been reported to be a potential virulence factor, and has been shown to be involved in a number of activities including induction of apoptosis, regulation of virus replication and modulation of the immune response. No function has yet been ascribed to N40, which represents an N-terminally deleted form of PB1. Previous studies on PB1-F2 function mainly used viruses genetically engineered to prevent PB1-F2 expression by mutation of the PB1-F2 start codon. However, ablation of the start codon was shown to increase the expression level of the downstream protein N40. In the present study, we generated recombinant A/WSN/33 viruses carrying different combinations of PB1-F2- and N40-knockout mutations. Overexpression of N40 in a PB1-F2-deficient background had a detrimental effect on virus growth in vitro and in vivo. However, ablation of PB1-F2 or N40 expression individually was not disadvantageous for the virus. Primer-extension analyses revealed an increase in vRNA production by viruses that overexpressed N40. Our data suggest that the observed attenuation of mutant viruses in vitro and in vivo results from these changes in transcription and replication.
甲型流感病毒基因组由 8 个负义 RNA 片段组成，编码多达 12 种蛋白。RNA 片段 2 编码三种蛋白，PB1、PB1-F2 和 N40，它们通过核糖体漏扫和重起始从同一条 mRNA 翻译而来。PB1 是三聚体病毒 RNA 聚合酶的一个亚基。PB1-F2 已被报道为一种潜在的毒力因子，已被证明参与多种活动，包括诱导细胞凋亡、调节病毒复制和调节免疫反应。N40 尚未赋予功能，它代表 PB1 的 N 端缺失形式。以前关于 PB1-F2 功能的研究主要使用通过突变 PB1-F2 起始密码子来阻止 PB1-F2 表达的遗传工程病毒。然而，去除起始密码子被证明会增加下游蛋白 N40 的表达水平。在本研究中，我们生成了携带 PB1-F2 和 N40 缺失突变不同组合的重组 A/WSN/33 病毒。在 PB1-F2 缺陷背景下过表达 N40 对病毒在体外和体内的生长有不利影响。然而，单独去除 PB1-F2 或 N40 的表达对病毒并没有不利影响。引物延伸分析显示，过表达 N40 的病毒 vRNA 产量增加。我们的数据表明，体外和体内观察到的突变病毒的衰减是由于转录和复制的这些变化所致。
