A Preliminary Study About Mechanism Of ISG15 In The Infection Of Mycobacterium Tuberculosis And Hepatitis C Virus

Backgroud:There is a co-infection phenomenon between Tuberculosis(TB)induced by Mycobacterium tuberculosis(M.tb)and hepatitis Ccaused by hepatitis C Virus(HCV),but their molecular mechanism is unknown.Recently,we have found that the Rv2645 gene in the RD13 region of M.tb H37Rv can significantly increase the expression of interferon(IFN)-?.The interferon-stimulated gene 15(ISG15)is a kind of IFN-?/?-induced Ubiquitin-like protein,participate in the anti-infection immune response.So we will investigate the effect of Rv2645 geneon the expression of ISG15 of the immune cells,and thenstudy the effect of ISG15 onthe infection of HCV in Huh7.5.1 cell.Recently,N6-methyladenosine(m6A)has become as a hotspot in epigenetic research,as one of the most common modification methods in eucaryon mRNA methylation modification.mRNA m6A is involved in regulating a variety of physiological activities of cells and plays an important role in gametogenesis,embryonic development and metabolism.Objective:In order to study the function and mechanism of ISG15 on the infection of M.tb and HCV,and to further explore the changes of m6A in HCV infection and the effect of ISG15 on the total RNA m6A in hepatocytes.These will provide a basis for elucidating the function of Rv2645 in virulent M.tbH37Rv,and provide an insight into the molecular mechanism of M.tb and HCV infections andan effective reference for the study of antiviral and anti-TB treatment,and the related treatment strategies.Methods:Non-virulent bovine tuberculosis Bacillus Calmette Guerin(BCG)carrying the Rv2645 gene(ie,rBCG)and BCG were used to infect mice and stimulate the murine macrophage line RAW264.7,respectively,RT-qPCR and Western Blot were used to detect the mRNA and protein levels of ISG15 in spleen CD4+T cells and RAW264.7 cells.The ISG15 eukaryotic expression plasmid and ISG15-shRNA(short hairpin RNA)were constructed and transfected into HCV-infected Huh7.5.1 cells,and then the expression of HCV mRNA,viral NS3 and Core protein were detected by RT-qPCR and Western Blot.At the same time,the m6A level of RNA in Huh7.5.1 cells after HCV infection was detected by mass spectrometry.The effects on m6A-related enzymes in cells infected with HCV and transfected with ISG15,were detected by RT-qPCR and Western Blot.Results:Compared to BCG treated group,the mRNA and protein levels of ISG15 were significantly increasedin the mouse spleen CD4+T cells and RAW264.7 cells after rBCG infection.The overexpression of ISG15 resulted in the increase of HCV mRNA,NS3 protein and Core proteinin Huh7.5.1 cells.The levels of ISG15 and HCV mRNA wereobviously decreased after ISG15 silencing.Further studies showed that the expressions of RNA methyltransferases(METTL3 and WTAP)were up-regulated in Huh7.5.1 cells after HCV infection,and the expression of demethylase(FTO)was down-regulated.Overexpression of ISG15 led to increased expressions of methyltransferases(METTL3 and WTAP)in Huh7.5.1 and HepG2 cells,while knowdown of ISG15 decraesed the expression levelof demethylaseFTO.These results is consistent with m6A semi-quantitative detection and m6A mass spectrometry.Conclution:M.tbRv2645 promotes the expression of ISG15 in themurine CD4+T cells and macrophages,whereas ISG15 promotes HCV proliferation in Huh7.5.1.In addition,HCV infection and overexpression of ISG15can promote mRNA m6A modification.This study provides a reference for elucidating the function of the Rv2645 gene of M.tbH37Rv and the mechanism of M.tb and HCV co-infection.It provides a new idea and research direction for studying the mechanism of chronic infection(such as liver cirrhosis,liver cancer)and the development of anti-HCV and anti-M.tb drugs.