National Avian Influenza Para-Reference Laboratory (Guangzhou), College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; Key Laboratory of Zoonosis, Ministry of Agriculture and Rural Affairs, Guangzhou, 510642, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangzhou, 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou, 510642, China.
National Avian Influenza Para-Reference Laboratory (Guangzhou), College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China.
Vet Microbiol. 2021 Mar;254:108985. doi: 10.1016/j.vetmic.2021.108985. Epub 2021 Jan 13.
The genome of influenza A virus is negative-sense and segmented RNA, which is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp) during the virus life cycle. The viral RdRp is thought to be an important host range and virulence determinant factor, and the 627 site of PB2 subunit is a highly acceptable key site of RdRp function. Besides, the function of RdRp is modulated by several host factors. Identification of the host factors interacting with RdRp is of great interest. Here, we tried to explore an effective method to study virus-host interaction by rescuing replication-competent recombinant influenza viruses carrying Strep tagged PB2. Subsequently, we tested several biological characteristics of recombinant viruses in cells and pathogenicity in mice. Then, we purified of protein complex of Strep tagged PB2 and host factors of interest from 293 T cells infected with recombinant viruses. After purification, we performed mass spectrometry to identify these proteins that interacting with PB2. We identified 57 host factors in total. Through Gene Ontology (GO) and Protein-Protein interaction (PPI) network analysis, we revealed the function and network of these proteins. In summary, we generated replication-competent recombinant influenza viruses by inserting a Strep-Tag into PB2 and purified host factors interacting with viral RdRp bearing a 627 K or 627E PB2. These proteins might function as host range and virulence determinants of influenza virus.
甲型流感病毒的基因组为负义、分段 RNA，在病毒生命周期中由病毒 RNA 依赖性 RNA 聚合酶（RdRp）转录和复制。病毒 RdRp 被认为是一个重要的宿主范围和毒力决定因素，而 PB2 亚基的 627 位是 RdRp 功能的高度可接受的关键位点。此外，RdRp 的功能受到几种宿主因素的调节。鉴定与 RdRp 相互作用的宿主因子具有重要意义。在这里，我们试图通过拯救携带 Strep 标记的 PB2 的复制型重组流感病毒来探索一种研究病毒-宿主相互作用的有效方法。随后，我们在细胞中测试了重组病毒的几种生物学特性和在小鼠中的致病性。然后，我们从感染重组病毒的 293T 细胞中纯化了 Strep 标记的 PB2 和感兴趣的宿主因子的蛋白复合物。纯化后，我们进行了质谱分析以鉴定与 PB2 相互作用的这些蛋白。我们总共鉴定了 57 个宿主因子。通过基因本体论（GO）和蛋白质-蛋白质相互作用（PPI）网络分析，我们揭示了这些蛋白的功能和网络。总之，我们通过在 PB2 中插入 Strep-Tag 生成了复制型重组流感病毒，并纯化了携带 627K 或 627E PB2 的病毒 RdRp 相互作用的宿主因子。这些蛋白可能作为流感病毒的宿主范围和毒力决定因素发挥作用。
