核蛋白(N)以其在负链RNA病毒基因组RNA的衣壳化中的功能而闻名,这导致核糖核蛋白的形成,作为病毒转录和复制的模板。然而,病毒感染过程中N蛋白在其他方面的功能尚不清楚。在这项研究中,蛇头囊泡病毒(SHVV)的N蛋白,一种鱼弹状病毒,被证明主要通过K63连接的泛素化被泛素化。我们确定了9个与SHVVN相互作用的宿主E3泛素连接酶,其中7种E3泛素连接酶促进N蛋白的泛素化。进一步调查显示,只有两种E3泛素连接酶,SiahE3泛素蛋白连接酶2(Siah2)和富含亮氨酸的重复序列和含有1(LRSAM1)的无菌α基序,介导的N蛋白的K63连接的泛素化。SHVV感染上调Siah2和LRSAM1的表达,维持了SHVVN的稳定性。Siah2或LRSAM1的过表达促进SHVV复制,而Siah2或LRSAM1的敲低抑制SHVV复制。Siah2或LRSAM1的连接酶结构域的缺失不会影响它们与SHVVN的相互作用,但会减少SHVVN和SHVV复制的K63连接的泛素化。总之,Siah2和LRSAM1介导SHVVN的K63连接的泛素化,以促进SHVV复制,这为负链RNA病毒的N蛋白的作用提供了新的见解。
目的:病毒蛋白的泛素化在病毒复制中起重要作用。然而,很少研究负链RNA病毒核蛋白(N)的泛素化。这项研究旨在研究鱼弹状病毒SHVV(蛇头囊泡病毒)的N蛋白的泛素化,鉴定相关宿主E3泛素连接酶,并确定SHVVN泛素化和宿主E3泛素连接酶在病毒复制中的作用。我们发现SHVVN主要通过K63连接的泛素化被泛素化,这是由宿主E3泛素连接酶Siah2(SiahE3泛素蛋白连接酶2)和LRSAM1(富含亮氨酸的重复序列和含有1的无菌α基序)介导的。数据表明,Siah2和LRSAM1被SHVV劫持以泛素化N蛋白用于病毒复制,它展示了用于药物设计的新型抗SHVV靶标。
Nucleoprotein (N) is well known for its function in the encapsidation of the genomic RNAs of negative-strand RNA viruses, which leads to the formation of ribonucleoproteins that serve as templates for viral transcription and replication. However, the function of the N protein in other aspects during viral infection is far from clear. In this study, the N protein of snakehead
vesiculovirus (SHVV), a kind of fish rhabdovirus, was proved to be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases that interacted with SHVV N, among which seven E3 ubiquitin ligases facilitated ubiquitination of the N protein. Further investigation revealed that only two E3 ubiquitin ligases, Siah E3 ubiquitin protein ligase 2 (Siah2) and leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1), mediated K63-linked ubiquitination of the N protein. SHVV infection upregulated the expression of Siah2 and LRSAM1, which maintained the stability of SHVV N. Besides, overexpression of Siah2 or LRSAM1 promoted SHVV replication, while knockdown of Siah2 or LRSAM1 inhibited SHVV replication. Deletion of the ligase domain of Siah2 or LRSAM1 did not affect their interactions with SHVV N but reduced the K63-linked ubiquitination of SHVV N and SHVV replication. In summary, Siah2 and LRSAM1 mediate K63-linked ubiquitination of SHVV N to facilitate SHVV replication, which provides novel insights into the role of the N proteins of negative-strand RNA viruses.
OBJECTIVE: Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead
vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design.