关键词: 口蹄疫病毒, 3′非编码RNA负链, 互作宿主蛋白, Ddx18, 2C

Abstract:

The aim of this study was to screen for the binding of the 3′UTR to the negative-strand RNA during replication of the foot and mouth disease virus (FMDV) negative-strand RNA. Firstly, we identified the host proteins that bind to the 3′UTR negative-strand by RNA pull down combined with mass spectrometry, and then we performed GO functional annotation analysis, KEGG pathway enrichment analysis, and screened of the interaction network on the identified proteins. We constructed eukaryotic plasmids as well as prokaryotic plasmids to express the proteins to validate the specificity of the interactions between the RNAs and the proteins. Secondly, we truncated the 3′UTR to explore the interaction region between RNA and protein. Finally, we used Co-IP to explore the interaction relationship between the interaction protein and viral protein. It was found that most of the proteins bound to the 3′UTR interactions were nucleolar proteins, among which the Ddx18 protein of the DEAD-box RNA helicase protein family was the focus of the study. By constructing the eukaryotic expression vector of this protein, we confirmed that Ddx18 did interact with the 3′UTR negative-strand using RNA pull down, RIP, and EMSA. A truncated 3′UTR negative-strand RNA was transcribed, and the RNA pull down assay was used to conclude that Ddx18 interact with the 3′UTR negative-strand. It does not depend on poly(U) and not interact with the truncated SL1 and SL2. Using Co-IP assay, it was demonstrated that 2C viral proteins can interact with Ddx18 proteins. In summary, the interactions between 3′UTR and Ddx18 protein may be based on spatial structure, whereas the 2C viral proteins bind to the Ddx18 protein and may indirectly bind to the 3′UTR and together form a replication complex that is involved in the replication of FMDV. By screening the host proteins that interact with the negative-strand of the 3′UTR, we can lay the experimental foundation for further investigating the mechanism related to FMDV replication and thus the related drug targets.

Key words: foot and mouth disease virus, the negative-strand of 3′UTR, interacting host proteins, Ddx18, 2C