Abstract
The rapid evolution of influenza virus makes antiviral drugs less effective, which is considered to be a major bottleneck in antiviral therapy. The key proteins in the host cells, which are related with the replication cycle of influenza virus, are regarded as potential drug targets due to their distinct advantage of lack of evolution and drug resistance. Cdc2-like kinase 1 (CLK1) in the host cells is responsible for alternative splicing of the M2 gene of influenza virus during influenza infection and replication. In this study, we carried out baculovirus-mediated expression and purification of CLK1 and established a reliable screening assay for CLK1 inhibitors. After a virtual screening of CLK1 inhibitors was performed, the activities of the selected compounds were evaluated. Finally, several compounds with strong inhibitory activity against CLK1 were discovered and their in vitro anti-influenza virus activities were validated using a cytopathic effect (CPE) reduction assay. The assay results showed that clypearin, corilagin, and pinosylvine were the most potential anti-influenza virus compounds as CLK1 inhibitors among the compounds tested. These findings will provide important information for new drug design and development in influenza treatment, and CLK1 may be a potent drug target for anti-influenza drug screening and discovery.
Highlights
Influenza A viruses are enveloped negative-sense, single-stranded segmented RNA viruses belonging to a genus of the Orthomyxoviridae family of viruses, which cause influenza in birds and some mammals
The total RNA extracted from Human Umbilical Vein Endothelial Cells (HUVEC) was used to synthesize cDNA, which was performed as the template in polymerase chain reaction (PCR) to produce full-length coding sequence (CDS) of Cdc2-like kinase 1 (CLK1) gene with BamH I and Not I at both ends, respectively
The recombined CLK1/pFastBac1 plasmid was transformed into DH5α E. coli strain and five ampicillin-resistant transformants were selected as follows: C-1, C-2, C-3, C-4 and C-5
Summary
Influenza A viruses are enveloped negative-sense, single-stranded segmented RNA viruses belonging to a genus of the Orthomyxoviridae family of viruses, which cause influenza in birds and some mammals. CLK1 play an important role in the regulation of alternative splicing of human genes through multisite phosphorylation of their RS serine/arginine-rich (SR) family of splicing factors [5] Some key technologies such as genome-wide RNA interference (RNAi) screening have accelerated the discovery and identification of host factors [6,7]. As one of these host cofactors which have been identified by RNAi required for influenza virus replication, CLK1 regulates alternative splicing of viral M1 pre-mRNA into M2 mRNA by phosphorylating the spliced factor SF2/ASF in infected primary normal human bronchial epithelial (NHBE) and human lung epithelial (A549) cellular assays. The in vitro anti-influenza virus activities of the CLK1 inhibitors were evaluated by the cytopathic effect assay to explain the possibility of using CLK1 as the antiviral drug target
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