RESUMO
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. IMPORTANCE: 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.
RESUMO
Snakehead vesiculovirus (SHVV) is a type of rhabdovirus that causes serious economic losses in snakehead fish culture in China. However, no specific antiviral drugs or vaccines are currently available for SHVV infection. In this study, 4D label-free ubiquitome analysis of SHVV-infected cells revealed dozens of ubiquitinated sites on the five SHVV proteins. We focused on investigating the ubiquitination of phosphoprotein (P), a viral polymerase co-factor involved in viral replication. SHVV-P was proved to be ubiquitinated via K63-linked ubiquitination at lysine 264 (K264). Overexpression of wild-type P, but not its K264R mutant, facilitated SHVV replication, indicating that K264 ubiquitination of the P protein is critical for SHVV replication. RNAi screening of 26 cellular E3 ubiquitin ligases identified five pro-viral factors for SHVV replication, including macrophage erythroblast attacher (MAEA), TNF receptor-associated factor 7 (TRAF7), and SH3 domain-containing ring finger protein 1 (SH3RF1), which interacted with and mediated ubiquitination of SHVV P. TRAF7 and SH3RF1, but not MAEA, mediated K63-linked ubiquitination of SHVV P, while only SH3RF1 mediated K264 ubiquitination of SHVV P. Besides, overexpression of SH3RF1 promoted SHVV replication and maintained the stability of SHVV P. In summary, SH3RF1 mediated K63-linked ubiquitination of SHVV P at K264 to facilitate SHVV replication, providing targets for developing anti-SHVV drugs and live-attenuated SHVV vaccines. Our study provides novel insights into the role of P protein in the replication of single-stranded, negative-sense RNA viruses.