Tingyu Lan1,2, Zhiqiang Wei3,4, Yulin He3,4, Song Wan2,4, Li Liu2,4, Bin Cheng3,4, Ruimin Li3,4, Hongxia Chen3,4, Guohua Liu3,4, Zhongji Meng5,6,7,8,9. 1. Postgraduate Training Basement of Jinzhou Medicical University, Taihe Hospital, Hubei University of Medicine, Shiyan, China. 2. Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, Shiyan, China. 3. Institute of Biomedical Research, Taihe Hospital, Hubei University of Medicine, No. 32, South Renmin Road, Shiyan, 442000, Hubei Province, China. 4. Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, China. 5. Postgraduate Training Basement of Jinzhou Medicical University, Taihe Hospital, Hubei University of Medicine, Shiyan, China. zhongji.meng@163.com. 6. Institute of Biomedical Research, Taihe Hospital, Hubei University of Medicine, No. 32, South Renmin Road, Shiyan, 442000, Hubei Province, China. zhongji.meng@163.com. 7. Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, Shiyan, China. zhongji.meng@163.com. 8. Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, China. zhongji.meng@163.com. 9. Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, 442000, China. zhongji.meng@163.com.
Abstract
BACKGROUND: Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. METHODS: msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-β, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-β were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. RESULTS: msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-β (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. CONCLUSIONS: msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.
BACKGROUND:Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. METHODS: msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-β, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-β were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. RESULTS: msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-β (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. CONCLUSIONS: msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.
Authors: Christine I Wooddell; Man-Fung Yuen; Henry Lik-Yuen Chan; Robert G Gish; Stephen A Locarnini; Deborah Chavez; Carlo Ferrari; Bruce D Given; James Hamilton; Steven B Kanner; Ching-Lung Lai; Johnson Y N Lau; Thomas Schluep; Zhao Xu; Robert E Lanford; David L Lewis Journal: Sci Transl Med Date: 2017-09-27 Impact factor: 17.956
Authors: Michael P Gantier; Stephen Tong; Mark A Behlke; Aaron T Irving; Martha Lappas; Ulrika W Nilsson; Eicke Latz; Nigel A J McMillan; Bryan R G Williams Journal: Mol Ther Date: 2010-02-02 Impact factor: 11.454
Authors: Alexandra Forsbach; Jean-Guy Nemorin; Carmen Montino; Christian Müller; Ulrike Samulowitz; Alain P Vicari; Marion Jurk; George K Mutwiri; Arthur M Krieg; Grayson B Lipford; Jörg Vollmer Journal: J Immunol Date: 2008-03-15 Impact factor: 5.422
Authors: Michael P Gantier; Stephen Tong; Mark A Behlke; Dakang Xu; Simon Phipps; Paul S Foster; Bryan R G Williams Journal: J Immunol Date: 2008-02-15 Impact factor: 5.422
Authors: Christine I Wooddell; David B Rozema; Markus Hossbach; Matthias John; Holly L Hamilton; Qili Chu; Julia O Hegge; Jason J Klein; Darren H Wakefield; Claudia E Oropeza; Jochen Deckert; Ingo Roehl; Kerstin Jahn-Hofmann; Philipp Hadwiger; Hans-Peter Vornlocher; Alan McLachlan; David L Lewis Journal: Mol Ther Date: 2013-02-26 Impact factor: 11.454