Characterization of host factors essential for

influenza A virus replication

Pages: 118
Language: English
Publication date: 30/11/-0001
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Dissertation Biologie
Influenza A virus carries eight negative single-stranded RNAs that encode only 14 viral proteins. Therefore, the complete replication processes are very dependent on cellular factors and processes. In the past years, there have been several genome-wide RNAi screens performed to identify essential host factors in influenza A virus replication. This approach is also useful to shed some light on potential target for influenza treatment. The genome-wide RNAi screen performed in our laboratory identifies Clk1 and eIF4A3 to be essential for influenza virus replication. Clk1 is a kinase that phosphorylates the RS domain of SR protein family, a key regulator in the alternative splicing process. The alternative splicing process is required in the processing of influenza A virus mRNAs to generate M2 ion-channel protein and NS2/NEP from segment 7 and 8, respectively. In this study, it is shown that Clk1 knockdown led to reduction of influenza A virus replication and increase of segment 7 mRNA splicing rate. The usage of an inhibitor with higher binding potency towards Clk1 also showed the same effect on segment 7 splicing regulation and additional effect on reduction of viral protein expression. Infection of clk1-/- mice or cells derived from the lung of clk1-/- mouse with influenza A virus resulted in reduction of the influenza virus replication, thus, supporting the relevance of Clk1 in influenza infection in vivo. Screening of newly-developed Clk inhibitors revealed several compounds which can effectively inhibit influenza A virus replication in A549 cells without any effect on cell viability, therefore, suggesting the potential use of Clk inhibitor for treatment of influenza infection. A further study was also performed on the role of eIF4A3, a protein that serves as the core component of the exon junction complex (EJC) and functions in multiple mRNA processing steps, i.e. splicing, mRNA export, non-sense mediated mRNA decay and translation. Here, it is shown that knockdown of eIF4A3 led to reduction of influenza A virus replication as well as viral M1, M2 and NP proteins expression, but did not affect viral M1 and NP mRNAs production. However, the M2 mRNA, which is produced from splicing process, was reduced in absence of eIF4A3, but only at later time points post infection. Study on the role of eIF4A3 in mRNA export demonstrated that eIF4A3 is required for the export of cellular mRNA and potentially be essential for viral NP mRNA export, but it is not required for the export of the viral M1 mRNA. In absence of eIF4A3, viral M1, M2 and NP mRNAs were found to be associated with less ribosomal complex, thus, suggesting the role of eIF4A3 in translation of viral mRNAs, which are mostly intronless and unspliced. Interaction study performed using lentiviralbased bi-fluorescence complementation assay revealed potential interaction of eIF4A3 with influenza PB1, PB2 and NS1 proteins. Taken together, this study increases the knowledge on the complexity of influenza virus mRNA splicing regulation and processing and it shed some lights on potential new target for influenza infection.
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