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Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein
Published on: April 21, 2015
MicroRNA-33a disturbs influenza A virus replication by targeting ARCN1 and inhibiting viral ribonucleoprotein
Yi Hu1, Liangzhen Jiang2, Wenbin Lai2
1Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, PR China.
Abstract:
In order to explore the roles of microRNA(s) [miRNA(s)] in the influenza A virus life cycle, we compared the miRNA profiles of 293T and HeLa cell lines, as influenza A virus can replicate efficiently in 293T cells but only poorly in HeLa cells. We analysed differentially expressed miRNAs and identified five, including miR-33a, that could disturb influenza A virus replication significantly. Using TargetScan analysis, we found that ARCN1 could be a potential target of miR-33a. To confirm whether miR-33a could truly target ARCN1, we generated a luciferase reporter for the ARCN1 3' untranslated region (UTR) and performed a luciferase assay. The data indicated that miR-33a could suppress the luciferase activity of the reporter for the ARCN1 3' UTR but not a reporter in which the predicted miR-33a targeting sites on ARCN1 3' UTR were mutated. We performed immunoblotting to confirm that miR-33a could downregulate the protein level of ARCN1. Consistently, the level of ARCN1 protein in HeLa cells was significantly lower than that in 293T cells. We also demonstrated that ectopic expression of ARCN1 could partially rescue the inhibitory effect of miR-33a on virus replication. Furthermore, we demonstrated that miR-33a could impede virus replication at the stage of virus internalization, which was similar to the pattern for knockdown of ARCN1, indicating that miR-33a inhibits influenza virus infection by suppressing ARCN1 expression. In addition, we found that miR-33a could also weaken the viral ribonucleoprotein activity in an ARCN1-independent manner. In conclusion, we found that miR-33a is a novel inhibitory factor for influenza A virus replication.
Insights
MicroRNA-33a (miR-33a) significantly inhibits influenza A virus replication by downregulating ARCN1 expression, impacting virus internalization. This discovery identifies miR-33a as a novel factor in controlling influenza virus infection.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Influenza A virus (IAV) replication efficiency varies between cell lines (e.g., 293T vs. HeLa).
- MicroRNAs (miRNAs) play crucial roles in regulating viral life cycles.
- Understanding miRNA-host interactions is key to developing antiviral strategies.
Purpose of the Study:
- To investigate the role of differentially expressed miRNAs in IAV replication.
- To identify specific miRNAs that can modulate IAV replication.
- To elucidate the molecular mechanism by which miR-33a affects IAV.
Main Methods:
- Comparative miRNA profiling of 293T and HeLa cells infected with IAV.
- Bioinformatic analysis (TargetScan) to predict miRNA targets.
- Luciferase reporter assays to validate miRNA-target interactions.
- Immunoblotting to assess protein level changes.
- Functional assays to evaluate the impact on viral replication and internalization.
Main Results:
- Five miRNAs, including miR-33a, were identified as significantly affecting IAV replication.
- ARCN1 was identified as a direct target of miR-33a, with miR-33a suppressing ARCN1 3' UTR activity.
- miR-33a was shown to downregulate ARCN1 protein levels.
- ARCN1 expression was lower in HeLa cells (poor IAV replication) compared to 293T cells (efficient replication).
- Ectopic ARCN1 expression partially rescued the inhibitory effect of miR-33a on IAV replication.
- miR-33a inhibited IAV replication at the internalization stage, similar to ARCN1 knockdown.
- miR-33a also affected viral ribonucleoprotein activity independently of ARCN1.
Conclusions:
- miR-33a is a novel inhibitory factor for influenza A virus replication.
- miR-33a inhibits IAV infection primarily by suppressing ARCN1 expression, affecting virus entry.
- miR-33a possesses additional mechanisms to impede viral replication, independent of ARCN1.
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