Original ArticleBta-miR-2411 attenuates bovine viral diarrhea virus replication via directly suppressing Pelota protein in Madin-Darby bovine kidney cells
Introduction
Small noncoding microRNAs (miRNAs) are a class of small noncoding RNAs that bind to complementary sequences in the 3′ untranslated region (UTR) of multiple target mRNAs and control their expression either by the translational repression or degradation of their target message (Iorio et al., 2005). Although miRNAs are tiny, they play important roles in signal transduction pathways, such as animal development (Alvarez-Garcia and Miska, 2005; Wienholds and Plasterk, 2005), cell proliferation (Johnson et al., 2007), autophagy (Comincini et al., 2013), apoptosis (Gong et al., 2013), and differentiation (Baumjohann and Ansel, 2013). To date, over 4000 miRNAs have been discovered in eukaryotes, including mammals, fungi and plants. More than 700 have been found and cloned in human genomes (Staszel et al., 2011). Currently, numerous research studies have indicated that most miRNA genes are transcribed by RNA polymerase II (Pol II), although some exceptions exist (Corcoran et al., 2009). The hairpin loop of the primary miRNA (pri-miRNA) is usually transcribed by the polymerase from the DNA sequence and capped with a specially modified nucleotide at the 5′ end, followed by polyadenylation with multiple adenosines (a poly(A) tail) and splicing, which generates a 72–80 nt precursor miRNA (pre-miRNA) (Cai et al., 2004; Lee et al., 2004). Pre-miRNAs are then exported to the cytoplasm by exporting 5 (XPO5)/CRM1 and are processed into mature miRNAs by Dicer1 and TRBP (Melo and Esteller, 2014).
Pelota, also known as Pelo in mammals and as Dom34 in yeast, is highly conserved from yeast to human and contains three eukaryotic translation termination factor 1 (eRF1) domains (Wu et al., 2014). Recent work has shown that the Pelota-Hbs1 complex is involved in the dissociation of stalled elongation complexes by promoting ribosomal subunit dissociation and the release of peptidyl-tRNA (Des Georges et al., 2013; Wu et al., 2014). Recognition of a stalled ribosomal complex in no-go decay (NGD) is followed by endonucleolytic cleavage of the mRNA upstream of the stalled ribosome in a Dom34-Hbs1-dependent manner (Roy and Jacobson, 2013). Indeed, deletion of Dom34 leads to at least partial stabilization of the stalled translation products (Shao et al., 2013). Moreover, recent research shows that the high level synthesis of Drosophila C virus (DCV) capsid proteins is limited as a result of Pelota mutation, suggesting that Pelota is a host factor which is required for high efficiency translation of viral capsids (Wu et al., 2014).
Bovine viral diarrhea virus (BVDV) is an enveloped, positive-sense, single-stranded RNA virus and belongs to the genus Pestivirus within the family Flaviviridae (Isken et al., 2014). BVDV causes numerous diseases in cattle, including diarrhea, mucosal disease, persistent infections, hemorrhagic syndrome, and reproductive and respiratory disorders, resulting in great economic losses to the cattle industry (Perdrizet et al., 1987).
In this report, prediction of miR-2411 targets led us to discover one of the miR-2411 response elements (MREs) in the 3′UTR of Pelota. Furthermore, we found that miR-2411 agomir transfection significantly inhibited Pelota expression and attenuated BVDV NADL replication. However, viral replication levels were reversed upon the Pelota rescuing experiment. Our findings underline the importance of miRNAs in controlling viral replication and introduce Pelota as a novel target for the general inhibition of viral replication.
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Cell lines and viral strain
The Madin-Darby bovine kidney (MDBK) and HEK-293T cell lines were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured according to the manufacturer’s instructions. Cell lines and culture media used for the cell cultures were tested and confirmed to be BVDV negative by reverse transcriptase polymerase chain reaction (PCR) assay (Weinstock et al., 2001).
The BVDV reference strain NADL was from National Institutes for Food and
MiR-2411 downregulates pelota expression by directly targeting its 3′UTR
We investigated the expression level of miR-2411 using Real-time PCR at different time intervals after BVDV infection. As shown in Fig. 1A, at 0, 4, 8 12, 24 and 36 h pi with BVDV NADL, the cells were harvested and subjected to miRNA quantification. The results demonstrated that BVDV NADL infection significantly increased miR-2411 expression levels after 8 h pi (Fig. 1A). Prediction of miRNA targets with the TargetScan algorithms revealed that Pelota 3′UTR contained a conserved miR-2411 7-mer
Discussion
To this point, many research studies have demonstrated that several miRNAs and miRNA clusters have different functions in pathogens in vivo and in vitro. For example, miR-29c overexpression significantly suppresses hepatitis B virus (HBV) DNA replication via targeting tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and downregulating its expression in hepatocellular carcinoma (HCC) cells, as well as inhibiting cell proliferation and inducing apoptosis (Wang et al., 2011). MiR-122 is a
Acknowledgments
This work was supported by Natural Science Foundation of China (Nos. 31502095, 31560328 and 31760742), China Postdoctoral Science Foundation (Nos. 2016M590988 and 2016M592868), Key Laboratory of Prevention and Control of Animal Disease of Xinjiang Corps (Nos. 2016BTDJ01 and 2016BTDJ02).
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These authors contributed equally to this work.