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. 2010 Sep;84(17):8849-60.
doi: 10.1128/JVI.00456-10. Epub 2010 Jun 16.

Cellular microRNAs inhibit replication of the H1N1 influenza A virus in infected cells

Liping Song  1 , He Liu, Shijuan Gao, Wei Jiang, Wenlin Huang
Affiliations

Cellular microRNAs inhibit replication of the H1N1 influenza A virus in infected cells

Liping Song et al. J Virol. 2010 Sep.

Abstract

MicroRNAs (miRNAs) are a class of noncoding RNAs of lengths ranging from 18 to 23 nucleotides (nt) that play critical roles in a wide variety of biological processes. There is a growing amount of evidence that miRNAs play critical roles in intricate host-pathogen interaction networks, but the involvement of miRNAs during influenza viral infection is unknown. To determine whether the cellular miRNAs play an important role in H1N1 influenza A viral infections, 3' untranslated region (UTR) reporter analysis was used to identify putative miRNA targets in the influenza virus genome, and virus proliferation analysis was used to detect the effect of the screened miRNAs on the replication of H1N1 influenza A virus (A/WSN/33) in MDCK cells. The results showed that miRNA 323 (miR-323), miR-491, and miR-654 inhibit replication of the H1N1 influenza A virus through binding to the PB1 gene. Moreover mutational analysis of the predicted miRNA binding sites showed that the three miRNAs bind to the same conserved region of the PB1 gene. Intriguingly, despite the fact that the miRNAs and PB1 mRNA binding sequences are not a perfect match, the miRNAs downregulate PB1 expression through mRNA degradation instead of translation repression. This is the first demonstration that cellular miRNAs regulate influenza viral replication by degradation of the viral gene. Our findings support the notion that any miRNA has antiviral potential, independent of its cellular function, and that the cellular miRNAs play an important role in the host, defending against virus infection.

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Figures

FIG. 1.
FIG. 1.
The PB1 gene may harbor potential binding sites for miRNAs. (a) Circular map of the pRL-TK vector. The IAV genes were inserted into the XbaI sites. (b) The relative luciferase activity from the pRL-TK reporter constructs carrying one of eight fragments of the H1N1 IAV and pGL3-control vector. (c) Schematic map of the PB1 genome. Numbers denote nucleotide positions spanned by fragments of the PB1 gene that were inserted into the 3′ UTR of the luciferase gene in pRL-TK. (d) The relative luciferase activity from pRL-TK reporter constructs carrying fragments from the dissection of the PB1 gene and pGL3-control vector. The relative luciferase activity from pRL-TK and pGL3-control vector was used as control.
FIG. 2.
FIG. 2.
miR-323, miR-491, and miR-654 inhibit expression of luciferase through binding to the PB1-5 gene fragment. (a) The relative luciferase activity from pRL-TK-PB1-5 and pGL3-control vector in 293T cells with overexpressing miRNAs. The control is the expression vector of C. elegans miR-239b. (b) Western blotting was used to detect the effects of miR-323, miR-491, and miR-654 on luciferase expression in 293T cells transfected with Flag-TK-PB1-5 or Flag-TK. (c) Western blotting was used to further detect the effects of miRNA inhibitors on luciferase expression in Flag-TK-PB1-5. The expressions of β-actin were analyzed as a control in Western blotting.
FIG. 3.
FIG. 3.
miR-323, miR-491, and miR-654 bind to the same region in the PB1 gene. (a) The sequences of miR-323, miR-491, miR-654, and PB1-5 mRNA. The same nucleotide sequence in miRNAs 5′-GUGG-3′ perfectly matches the 5′-CCAC-3′ sequence in the PB1 mRNA. (b) The model of hybridization between the miRNAs and PB1-5 mRNA were predicted using RNAHybrid software. (c) The mutant nucleotide sequence of pRL-TK-PB1-5 and sequencing analysis. (d) The mutant nucleotide sequences of the miRNAs. The mutant 5′-UUCC-3′ sequences in ΔmiRNAs perfectly match the 5′-GGAA-3′ sequence in ΔpRL-TK-PB1-5 mRNA. (e) Luciferase assay to analyze the importance of binding sites of miRNAs in the PB1 gene for miRNAs to inhibit the relative luciferase activity from pRL-TK-PB1-5 and pGL3-control vector.
FIG. 4.
FIG. 4.
miR-323, miR-491, and miR-654 downregulate PB1 expression through mRNA degradation in MDCK cells. (a) The mutant PB1 gene. The nucleotide sequence, but not the amino acid sequence, of the PB1 gene was altered. (b) Western blotting was used to detect the effects of miR-323, miR-491, and miR-654 on PB1 expression or mutant PB1 expression in MDCK cells. Lanes 1 to 5 are the samples in MDCK cells and MDCK cells overexpressing C. elegans miR-239b, miR-323, miR-491, and miR-654, respectively. (c) Real-time PCR was used to detect the expression level of the PB1 mRNA and mutant PB1 mRNA in MDCK cells and MDCK cells overexpressing C. elegans miR-239b, siRNA-PB1, miR-323, miR-491, and miR-654. The expression vectors of C. elegans miR-239b and siRNA-PB1 were used as a miRNA negative control and positive control, respectively. *, the results were significantly different (P < 0.01).
FIG. 5.
FIG. 5.
The miRNAs inhibit the replication of H1N1 IAV through binding to the PB1 gene in MDCK cells. (a) The HA values were used to assess the effects of miRNAs on replication of H1N1 IAV in MDCK cells; the HA values were determined in triplicate at different time points. (b) Fluorescence microscopy was used to verify the effect of miRNAs on replication of H1N1 IAV in MDCK cells. (c) Real-time PCR was used to detect the effects of miRNA expression vectors and miRNA inhibitors on the relative expressions of the PB1 gene in virus-infected MDCK cells. (d) The HA values were used to determine the effect miRNAs on replication of mutant H1N1 IAV in MDCK cells; the HA values were determined in triplicate at different time points. The expression vector of C. elegans miR-239b was used as negative control.
FIG. 6.
FIG. 6.
The relative expression levels of miR-323, miR-491, and miR-654 in different cell lines. The relative expression of miR-323 was 10 × 2(CT1 −CT2). The relative expression of miR-491 was 100 × 2(CT1 − CT2). The relative expression of miR-654 was 1,000 × 2(CT1 −CT2). CT1, the cycle threshold value of U6; CT2: the CT value of miRNAs.
FIG. 7.
FIG. 7.
Endogenous miR-323, miR-491, and miR-654 inhibit the replication of HIN1 IAV in MDCK cells. *, results were significantly different (P < 0.01).
FIG. 8.
FIG. 8.
The relative expression of miR-323, miR-491, and miR-654 in MDCK cells with or without H1N1 IAV infection. x axis represents the different infection time points.
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References

    1. Andersson, M. G., P. C. Haasnoot, N. Xu, S. Berenjian, B. Berkhout, and G. Akusjarvi. 2005. Suppression of RNA interference by adenovirus virus-associated RNA. J. Virol. 79:9556-9565. - PMC - PubMed
    1. Aukerman, M. J., and H. Sakai. 2003. Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15:2730-2741. - PMC - PubMed
    1. Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-297. - PubMed
    1. Bennasser, Y., S. Y. Le, M. Benkirane, and K. T. Jeang. 2005. Evidence that HIV-1 encodes an siRNA and a suppressor of RNA silencing. Immunity 22:607-619. - PubMed
    1. Bentwich, I. 2005. Prediction and validation of microRNAs and their targets. FEBS Lett. 579:5904-5910. - PubMed

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