Yuan Honggen, Luo Yun, Zou Jiahui, Zhang Junmei, Zhang Jinhua, Cao Gang, Cao Shengbo, Chen Huanchun, Song Yunfeng
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
J Virol. 2024 Dec 17;98(12):e0029724. doi: 10.1128/jvi.00297-24. Epub 2024 Nov 5.
A cellular protein, non-POU-domain-containing octamer binding protein (NONO), bound to the replication complex of Japanese encephalitis virus (JEV) by directly interacting with the viral 3' UTR RNA and NS3 protein. These interactions were also identified in West Nile virus (WNV) and Zika virus (ZIKV). The infection of JEV or the expression of JEV NS3 protein in cells could induce relocation of NONO protein from the nucleus to the cytoplasm. In JEV-infected cells, the NS3, NS5, and viral RNA could be concurrently detected in the immunoprecipitation by the NONO-specific antibody, suggesting that NONO could integrate into the replication complex of JEV. Further results of co-immunoprecipitation assays showed that NONO protein interacted with NS3 helicase domains 1 and 2 by its two RNA recognize motifs (RRMs). The knockdown and knockout of NONO in cells could significantly reduce the replication of JEV and ZIKV but had no effect on the replication of vesicular stomatitis virus (VSV). The effect of NONO protein on JEV proliferation occurred during the replication stage, rather than the attachment and entry stages. The level of viral positive-strand RNA in NONO knockout cells was significantly reduced than that in wild-type cells at 12-48 h post-JEV infection. However, the level of negative-strand virus RNA had no difference between NONO knockout and wild-type cells at 12-24 h post-infection. In summary, our study identified a cellular protein that bound to the replication complex of flavivirus and facilitated the synthesis of positive-strand RNA.IMPORTANCEOver half of the world's population is at risk of flaviviruses infection, posing a serious global health concern. To date, there are no antiviral drugs or treatments for the severe symptoms caused by the infection of flaviviruses. Some cellular proteins could participate in the replication of virus, and these cellular proteins were also ideal targets in antiviral strategy. Here, we identified cellular NONO protein was recruited by flavivirus NS3 protein to the cytoplasm, serving as a "scaffold" for viral replication complex. Our findings also revealed that NONO protein was critical for flavivirus positive-strand RNA synthesis. Specific areas where NONO interacted with flavivirus NS3 proteins and viral UTRs have also been identified. These results propose a new mechanism for cellular protein to participate in flavivirus replication and also raise a new potential anti-flavivirus strategy.
一种细胞蛋白，即不含POU结构域的八聚体结合蛋白（NONO），通过与日本脑炎病毒（JEV）的3'UTR RNA和NS3蛋白直接相互作用，与该病毒的复制复合体结合。在西尼罗河病毒（WNV）和寨卡病毒（ZIKV）中也发现了这些相互作用。JEV感染细胞或在细胞中表达JEV NS3蛋白可诱导NONO蛋白从细胞核重新定位到细胞质。在JEV感染的细胞中，通过NONO特异性抗体进行免疫沉淀可同时检测到NS3、NS5和病毒RNA，这表明NONO可整合到JEV的复制复合体中。免疫共沉淀试验的进一步结果表明，NONO蛋白通过其两个RNA识别基序（RRMs）与NS3解旋酶结构域1和2相互作用。在细胞中敲低和敲除NONO可显著降低JEV和ZIKV的复制，但对水疱性口炎病毒（VSV）的复制没有影响。NONO蛋白对JEV增殖的影响发生在复制阶段，而非附着和进入阶段。在JEV感染后12至48小时，NONO敲除细胞中的病毒正链RNA水平显著低于野生型细胞。然而，在感染后12至24小时，NONO敲除细胞和野生型细胞之间的负链病毒RNA水平没有差异。总之，我们的研究鉴定出一种与黄病毒复制复合体结合并促进正链RNA合成的细胞蛋白。重要性世界上超过一半的人口面临黄病毒感染风险，这是一个严重的全球健康问题。迄今为止，尚无针对黄病毒感染所致严重症状有效的抗病毒药物或治疗方法。一些细胞蛋白可参与病毒复制，这些细胞蛋白也是抗病毒策略中的理想靶点。在此，我们发现黄病毒NS3蛋白将细胞NONO蛋白募集到细胞质中，作为病毒复制复合体的 “支架”。我们的研究结果还表明，NONO蛋白对黄病毒正链RNA合成至关重要。还确定了NONO与黄病毒NS3蛋白和病毒UTR相互作用的特定区域。这些结果提出了细胞蛋白参与黄病毒复制的新机制，也提出了一种新的潜在抗黄病毒策略。
