Department of Pathogen Biology, College of Basic Medical Sciences, China Medical University, Shenyang 110122, PR China.
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
J Gen Virol. 2020 Dec;101(12):1242-1250. doi: 10.1099/jgv.0.001503. Epub 2020 Oct 5.
Post-translational modification plays a critical role in viral replication. Previously we reported that neddylation of PB2 of influenza A virus (IAV) can inhibit viral replication. However, we found that NEDD8 overexpression can still inhibit the replication of PB2 K699R mutant viruses, implying that other viral protein(s) can be neddylated. In this study, we revealed that M1 of IAV can also be modified by NEDD8. We found that the E3 ligase HDM2 significantly promotes M1 neddylation. Furthermore, we identified M1 K187 as the major neddylation site. We generated an IAV M1 K187R mutant (WSN-M1 K187R) and compared the growth of wild-type and mutant viruses in Madin-Darby canine kidney (MDCK) cells. The data showed that the replication of WSN-M1 K187R was more efficient than that of wild-type WSN. More importantly, we observed that overexpression of NEDD8 inhibited the replication of the wild-type WSN more effectively than that of WSN-M1 K187R. In addition, we found that the neddylation-deficient M1 mutant (M1 K187R) had a longer half-life than that of wild-type M1, indicating that the neddylation of M1 reduces stability. Then we performed a viral infection assay and found that WSN-M1 K187R exhibited greater virulence in mice than wild-type WSN, suggesting that the neddylation of M1 reduced IAV replication . In conclusion, we uncovered that neddylation of M1 by HDM2 negatively regulates the stability of M1, which in turn inhibits viral replication.
翻译后修饰在病毒复制中起着关键作用。此前我们报道甲型流感病毒（IAV）的PB2的NEDD8化可抑制病毒复制。然而，我们发现NEDD8过表达仍可抑制PB2 K699R突变病毒的复制，这意味着其他病毒蛋白也可被NEDD8化。在本研究中，我们揭示IAV的M1也可被NEDD8修饰。我们发现E3连接酶HDM2显著促进M1的NEDD8化。此外，我们确定M1的K187为主要的NEDD8化位点。我们构建了IAV M1 K187R突变体（WSN-M1 K187R），并比较了野生型和突变型病毒在犬肾传代细胞（MDCK）中的生长情况。数据显示，WSN-M1 K187R的复制比野生型WSN更有效。更重要的是，我们观察到NEDD8过表达对野生型WSN复制的抑制作用比对WSN-M1 K187R更有效。此外，我们发现NEDD8化缺陷的M1突变体（M1 K187R）的半衰期比野生型M1更长，这表明M1的NEDD8化降低了稳定性。然后我们进行了病毒感染试验，发现WSN-M1 K187R在小鼠中的毒力比野生型WSN更强，这表明M1的NEDD8化降低了IAV的复制。总之，我们发现HDM对M1的NEDD8化负向调节M1的稳定性，进而抑制病毒复制。
