MicroRNA Profile Analysis Of F81Cell Line Before And After Infection With Mink Enteritis Virus And Related MicroRNA Functional Characterization

Interaction between virus and host, as a scientific issue, has caused great attention of virologist. Currently, although some mechanisms about their interaction have been elucidated, little is known about a number of interaction networks involving microRNA (miRNA). MiRNAs are endogenous small noncoding RNAs of length18-23nucleotides (nt), which mediate post-transcriptional regulation of target genes through mRNA degradation or translational repression and play critical roles in many biological processes including cell proliferation, differentiation and haematopoiesis. Recent studies have also noted the role of miRNAs as modulators in host-pathogen interaction networks, such as foaming virus, hepatitis C virus, porcine reproductive and respiratory syndrome virus and influenza virus.Therefore, a simple parvovirus-mink enteritis virus (MEV) was selected as a model to explore the interaction networks. MEV, a subspecies of the feline parvovirus (FPV) and belonging to the family Parvoviridae, is one of the most important viral pathogens in the mink industry, which causes a highly infectious acute disease and has a high rate of morbidity and mortality in mink, resulting in huge economic losses in the worldwide. MEV is a single-stranded negative sense DNA virus with a genome comprised mainly of2ORFs, one coding for none-structural protein (NS) and another coding for capsid protein.To study the involvement of miRNAs in the MEV infection process, we used Illumina’s ultra high throughput approach to sequencing miRNA libraries from the feline kidney (F81) cell line before and after infection with MEV and identified miRNAs involved in MEV infection.Using this bioinformatics approach we identified196known mammalian miRNA orthologs belonging to152miRNA families in F81cells. Additionally,97miRNA*s of these miRNAs were also detected. Besides known miRNAs,384and398novel miRNA precursor candidates were identified from uninfected F81cells and MEV-infected cells respectively that have not been reported in other mammals. The majority (12of16) of randomly selected miRNAs expression profiles by qRT-PCR were consistent with those by deep sequencing. After target prediction,8miRNAs appeared to target the MEV mRNA coding region; and6to target the3’untranslated region (UTR) of MEV-specific receptor transferring receptor (TfR) mRNAs.Among the8miRNAs predicted to target MEV mRNA coding region, miR-181b showing comparatively higher expression level was selected. The potential target of miR-181b in NS1mRNA was highly conserved in different parvoviruses. After transfection with synthetic miRNA mimics and inhibitors, detection the level of viral titer, viral genomic DNA, the quantity of infected cells, cytopathic effect and immunostaining were carried out. The results showed that miR-181b mimics inhibited MEV production and miR-181b inhibitors increased it in the late virus infection. Dual-luciferase reporter assay validated the authenticity of miR-181b potential target. Western blot assay of NS1protein showed the inhibiton of miR-181b in NS1expression. Through detection the effect of miR-181b on the potential target mutated MEV, we confirmed that miR-181b inhibited MEV production through the potential target. Argonaute2(Ago2) co-immunoprecipitation demonstrated that miR-181b bound NS1mRNA in RNA-induced silencing complex (RISC). In the meantime, MEV infection led to the change of miRNA expression including miR-181b. Experiment results presented here show that cellular miR-181b in F81cells inhibits replication of MEV by targeting its NS1mRNA coding region resulting in NS1translational repression.MEV infection is dependent on induction of transferrin receptor (TfR). By homologous alignment, TfR mRNA3’UTR was cloned and sequenced. From the6predicted miRNAs, qPCR and western blot assay revealed that miR-320a and miR-140inhibited TfR expression and flow cytometric assay also verified the results. MEV infection led to gradually increase of the2miRNAs and decrease of TfR. Dual-luciferase reporter assay validated the authenticity of the2miRNA potential targets. Ago2co-immunoprecipitation demonstrated that the2miRNAs bound TfR mRNA in RISC. Quantification of MEV genomic DNA level at the indicated times validated the inhibition of the2miRNAs in MEV infection. Additionally, the2miRNAs played the inhibiton roles on TfR and MEV in a synergistic manner. Experiment results also show that miR-320a and miR-140inhibit MEV entry into F81cells by down-regulating its receptor TfR through targeting the3’UTR of TfR mRNA.This is the first time to construct the miRNA base of F81cells before and after MEV infection and to describe that cellular miR-181b inhibits replication of MEV by targeting its NS1mRNA coding region resulting in NS1translational repression; and miR-320a and miR-140inhibit MEV entry into F81cells by down-regulating its receptor TfR through targeting the3’UTR of TfR mRNA. These results provide further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies.