MicroRNA miR-214 inhibits snakehead vesiculovirus replication by targeting the coding regions of viral N and P

Abstract

Snakeheadvesiculovirus (SHVV), a new member of the family Rhabdoviridae, has caused enormous economic losses in snakehead fish culture during the past years in China; however, little is known about the molecular mechanisms of its pathogenicity. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in virus infection. In this study, we identified that SHVV infection downregulated miR-214 in striped snakehead (SSN-1) cells in a time- and dose-dependent manner. Notably, transfecting SSN-1 cells with miR-214 mimic significantly inhibitedSHVV replication, whereas miR-214 inhibitor promoted it, suggesting that miR-214 acted as a negative regulator of SHVV replication. Our study further demonstrated that N and P of SHVV were the target genes of miR-214. Over-expression of P, but not N, inhibited IFN-α production in SHVV-infected cells, which could be restored by over-expression of miR-214. Taken together, these results suggest that miR-214 is downregulated during SHVV infection, and the downregulated miR-214 in turn increased N and P expression and decreased IFN-α production, thus facilitating SHVV replication. This study provides a better understanding of the molecular mechanisms on the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.

MicroRNA miR-214 Inhibits Snakehead Vesiculovirus Replication by Promoting IFN-α Expression via Targeting Host Adenosine 5′-Monophosphate-Activated Protein Kinase

“…The miR-214 mimic, miR-214 inhibitor, negative control (NC) mimic, and NC inhibitor were purchased from GenePharma (Shanghai, China). Their sequences were previously described ( 15 ). Two siRNAs for AMPK (accession number: MF989224) were synthesized from GenePharma (Shanghai, China).…”

“…The dual-luciferase reporter assay was performed as described previously ( 15 ). In brief, SSN-1 cells were co-transfected with NC mimic, miR-214 mimic, NC inhibitor, or miR-214 inhibitor, together with the luciferase reporter plasmids using TransIntroTM EL Transfection Reagent (TransGen Biotech, China).…”

“…Virus infection and titration experiments were performed as previously described ( 15 ). In brief, SSN-1 cells were incubated with SHVV for 2 h, the inoculum was then removed and the cells were washed twice with PBS followed by adding MEM medium with 5% FBS.…”

“…Total RNAs were extracted from cells with TRIzol reagent (Invitrogen) according to manufacturer’s instructions. The detection of viral mRNA, host IFN-α (accession number: MF989225) mRNA, and miR-214 was performed by qRT-PCR as previously described ( 15 ). Two sets of data were normalized using the 2 −ΔΔCt method.…”

“…Its genome is an ~11 kb negative-sense RNA molecule that encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase protein (L) ( 12 ). Our previous study has revealed that SHVV infection downregulated miR-214 ( 14 ), and in turn, miR-214 could inhibit SHVV production by targeting viral N and P ( 15 ). However, it is unclear whether miR-214 can regulate SHVV replication via targeting host factors that are required for SHVV replication.…”