| Background:Seasonal influenza caused by influenza A and B virus infections results in a heavy global health economic burden.Although the spread of seasonal influenza viruses declined during 2020-2021 following the emergence of SARS-CoV-2,infection rates are trending back up in 2022.Against influenza viruses,vaccine prophylaxis is the primary option.However,the high error rate of RNA-dependent RNA polymerase and the recombination of RNA fragments in co-infections have provided influenza A viruses with the ability to evolve,making vaccines unable to inhibit virus transmission more efficiently due to "timeliness" limitations.In addition to vaccines,antiviral drugs play a critical role in the prevention and treatment of influenza virus infections.Two drugs currently approved by the FDA for the treatment of influenza are M2 ion channel inhibitors and NA inhibitors.However,influenza A viruses are widely resistant to M2 ion channel inhibitors,and researchers are looking at the development of new target antiviral drugs.Clathrin-mediated endocytosis is one of the ways for influenza virus to enter the host cell.Cargo is packaged into vesicles by clathrin,which are the basis for intracellular neurotransmission,signal transduction and regulation of many plasma membrane activities.UCM05,a nalog of Ellagitannins,as well as a fatty acid synthase inhibitor,which was found to inhibit the proliferation of human breast cancer cell lines.In our preliminary cellular experiments,we performed protein-level-based antiviral screening and discovered that UCM05 could resist influenza A virus,which may provide new ideas for the development of novel anti-influenza virus drugs.Objectives:In this paper,we investigated the mechanism through experiments on the inhibition of influenza A virus activity by the small molecule compound UCM05,with a view to finding new target drugs against influenza virus and providing new ideas for the development of novel anti-influenza virus drugs.Methods:1.The cytotoxicity of UCM05 on MDCK,A549 and Beas-2B cells and its antiviral activity by MTT assay and the protection of cells by CPE assay.2.To detect the inhibitory effect of UCM05 on influenza A virus at protein and mRNA levels as determined by Western blotting assay,qRT-PCR assay and indirect immunofluorescence assay.3.Detection of the phase of action of UCM05 against influenza A virus by H5N1 pseudovirus inhibition assay,hemagglutination inhibition assay,NA inhibition assay,and mini-replicon assay.4.Detecting the target of influenza A virus inhibition by UCM05 through pre-fusion inhibition assay,adsorption inhibition assay,transferrin and caveolin-1 uptake inhibition assay,and immunoconfocal assay.5.To detect the anti-influenza A virus effect of UCM05 in vivo.6.To detect the antiviral activity of UCM05 against ZIKV,HSV-1/2,HCoV-OC43 and SARS-CoV-2 pseudovirus,by MTT assay,Western blotting assay,qRT-PCR assay,etc.Results:1.UCM05 had inhibitory effect on MDCK,A549 and Beas-2B cells against influenza A virus and reduces the viral expression of the progeny.2.UCM05 inhibited the entry of pseudovirus,but had neither inhibitory effect on hemagglutination nor inhibitory effect on neuraminidase,and no significant inhibition on RNA polymerase.The strongest suppressive activity at 0-2 h in virusinfected cells.3.UCM05 could inhibit the uptake of transferrin and has no significant inhibitory effect on caveolin-1.4.UCM05 ameliorated the symptoms of influenza virus-induced viral pneumonia in vivo and reduced the levels of relevant inflammatory factors in serum and lung tissues of mice.5.UCM05 showed some inhibitory activity against ZIKV,HSV 1/2,and HCoVOC43,as well as against and SARS-CoV-2 pseudovirus entry.Conclusions:UCM05 was able to inhibit influenza A virus infection in vitro and in vivo.It mainly acts on the entry phase of virus invasion into the host and inhibits virus proliferation by inhibiting the clathrin-dependent endocytosis pathway,with no significant inhibitory effect on the caveolin-1.Meanwhile,UCM05 has potential broadspectrum antiviral activity. |
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