Email this article Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
- Authors: Brezgin S.A.1,2, Kostyusheva A.P.1, Simirsky V.N.3, Volchkova E.V.4, Chistyakov D.S.4, Kostyushev D.S.1, Chulanov V.P.1,4
-
Affiliations:
- Central Research Institute of Epidemiology, Rospotrebnadzor
- Institute of Immunology FMBA
- Koltzov Institute of Developmental Biology of Russian Academy of Sciences
- I.M. Sechenov Moscow State Medical University
- Issue: Vol 9, No 3-4 (2019)
- Pages: 476-484
- Section: ORIGINAL ARTICLES
- URL: https://iimmun.ru/iimm/article/view/922
- DOI: https://doi.org/10.15789/2220-7619-2019-3-4-476-484
- ID: 922
Cite item
Full Text
Abstract
Chronic hepatitis B is a severe liver disease associated with persistent infection with hepatitis B virus. According to recent estimations, 250 million people in the world are chronically infected, including 3 million chronically infected people in Russia. Antiviral therapeutics (nucleos(t)ide analogues and PEGylated interferon) suppress viral transcription and replication, but do not eliminate the virus from infected cells. The key reason for HBV persistency is a stable form of the viral genome (covalently closed circular DNA, cccDNA) that exists as a minichromosome protected from novel cccDNA-targeting therapeutics. Novel therapeutic approaches aimed at elimination or inactivation of cccDNA are urgently needed. CRISPR/Cas9 systems induce double strand breaks in target sites of DNA sequences. Experiments with CRISPR/Cas9 demonstrated high antiviral activity and efficient cleavage of cccDNA, but a small part of cccDNA pool remains intact. One of the main reasons of incomplete cccDNA elimination might be the structural organization of cccDNA, which persists in a heterochromatinized, very compacted form and is not be accessible to CRISPR/Cas9 systems. Viral protein HBx unwinds cccDNA and regulates cccDNA epigenetically by recruiting transcription-remodeling factors. In this work, we analyzed effects of CRISPR/Cas9 in combination with an HBxencoding plasmid or plasmids encoding mutant forms of HBx (HBxMut, which does not interact with pro-apoptotic factors Bcl-2 и Bcl-xL, and HBxNesm is localized exclusively in the nucleus and does not generate reactive oxygen species and double strand breaks in the genome). We showed that HBx improves CRISPR/Cas9 efficiency, decreasing pregenomic RNA transcription level over 98%. Moreover, we analyzed optimal ratios of plasmids encoding CRISPR/ Cas9 and HBx proteins for better antiviral efficacy. Furthermore, we discovered that HBx proteins do not have an effect on proliferation and viability of the transfected cells. In conclusion, CRISPR/Cas9 with HBx proteins exhibit high antiviral effect.
About the authors
S. A. Brezgin
Central Research Institute of Epidemiology, Rospotrebnadzor;Institute of Immunology FMBA
Email: sb@rcvh.ru
Junior Researcher, Laboratory of Viral Hepatitis;
PhD Student, Laboratory of Clinical Pharmacology No. 73,
Moscow
Russian FederationA. P. Kostyusheva
Central Research Institute of Epidemiology, Rospotrebnadzor
Author for correspondence.
Email: ak@rcvh.ru
Junior Researcher, Laboratory of Viral Hepatitis,
111123, Moscow, Novogireevskaya str., 3A
Russian FederationV. N. Simirsky
Koltzov Institute of Developmental Biology of Russian Academy of Sciences
Email: simir@mail.ru
PhD (Biology), Senior Researcher,
Moscow
E. V. Volchkova
I.M. Sechenov Moscow State Medical University
Email: fake@neicon.ru
PhD, MD (Medicine), Professor, Professor of the Department of Infectious Diseases, Head of the Department of Infectious Diseases on Faculty of Preventive Medicine,
Moscow
Russian FederationD. S. Chistyakov
I.M. Sechenov Moscow State Medical University
Email: fake@neicon.ru
Laboratory Technician,
Moscow
D. S. Kostyushev
Central Research Institute of Epidemiology, Rospotrebnadzor
Email: dchistakoff@gmail.com
Researcher,
Moscow
V. P. Chulanov
Central Research Institute of Epidemiology, Rospotrebnadzor;I.M. Sechenov Moscow State Medical University
Email: vladimir.chulanov@rcvh.ru
PhD, MD (Medicine), Head of the Laboratory of Viral Hepatitis;
Professor of the Department of Infectious Diseases, Faculty of Preventive Medicine,
Moscow
References
- Allweiss L., Dandri M. The Role of cccDNA in HBV Maintenance. Viruses, 2017, vol. 9, no. 6: 156. doi: 10.3390/v9060156
- Belloni L., Pollicino T., De Nicola F., Guerrieri F., Raffa G., Fanciulli M., Levrero M. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc. Natl. Acad. Sci. USA, 2009, vol. 106, no. 47, pp. 19975– 19979. doi: 10.1073/pnas.0908365106
- Bock C.T., Schwinn S., Locarnini S., Fyfe J., Manns M.P., Trautwein C., Zentgraf H. Structural organization of the hepatitis B virus minichromosome. J. Mol. Biol., 2001, vol. 307, no. 1, pp. 183–196. doi: 10.1006/jmbi.2000.4481
- Cha M.-Y., Kim C.-M., Park Y.-M., Ryu W.-S. Hepatitis B virus X protein is essential for the activation of Wnt/beta-catenin signaling in hepatoma cells. Hepatology, 2004, vol. 39, no. 6, pp. 1683–1693. doi: 10.1002/hep.20245
- Dong C., Qu L., Wang H., Wei L., Dong Y., Xiong S. Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication. Antiviral Res., 2015, vol. 118, pp. 110–117. doi: 10.1016/j.antiviral.2015.03.015
- Forgues M., Marrogi A.J., Spillare E.A., Wu C.G., Yang Q., Yoshida M., Wang X.W. Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway. J. Biol. Chem., 2001, vol. 276, no. 25, pp. 22797–22803. doi: 10.1074/jbc.M101259200
- Ganem D., Prince A.M. Hepatitis B virus infection — natural history and clinical consequences. N. Engl. J. Med., 2004, vol. 350, no. 11, pp. 1118–1129. doi: 10.1056/NEJMra031087
- Geng X., Huang C., Qin Y., McCombs J.E., Yuan Q., Harry B.L., Xue D. Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction. Proc. Natl. Acad. Sci. USA, 2012, vol. 109, no. 45, pp. 18471–18476. doi: 10.1073/pnas.1204668109
- Kim S., Lee H.-S., Ji J.-H., Cho M.-Y., Yoo Y.-S., Park Y.-Y., Cho H. Nuclear expression of hepatitis B virus X protein is associated with recurrence of early-stage hepatocellular carcinomas: role of viral protein in tumor recurrence. J. Pathol. Transl. Med., 2016, vol. 50, no. 3, pp. 181–189. doi: 10.4132/jptm.2016.03.18
- Konerman M.A., Lok A.S. Interferon treatment for hepatitis B. Clin. Liver Dis., 2016, vol. 20, no. 4, pp. 645–665. doi: 10.1016/j.cld.2016.06.002
- Liu Y., Zhao M., Gong M., Xu Y., Xie C., Deng H., Wang Z. Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus. Antiviral Res., 2018, vol. 152, pp. 58–67. doi: 10.1016/j.antiviral.2018.02.011
- Lucifora J., Xia Y., Reisinger F., Zhang K., Stadler D., Cheng X., Volz T. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science, 2014, vol. 343, no. 6176, pp. 1221–1228.
- Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut, 2015, vol. 64, no. 12, pp. 1972–1984. doi: 10.1136/gutjnl-2015-309809
- Ramanan V., Shlomai A., Cox D.B.T., Schwartz R.E., Michailidis E., Bhatta A., Bhatia S.N. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci. Rep., 2015, vol. 5: 10833. doi: 10.1038/srep10833
- Seeger C., Sohn J.A. Targeting hepatitis B virus with CRISPR/Cas9. Mol. Ther. Nucleic Acids, 2014, vol. 3: e216. doi: 10.1038/mtna.2014.68
- Seeger C., Sohn J.A. Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA. Mol. Ther., 2016, vol. 24, no. 7, pp. 1258–1266. doi: 10.1038/mt.2016.94
- Shi H., Lu L., Zhang N.-P., Zhang S.-C., Shen X.-Z. Effect of interferon-gamma and tumor necrosis factor-alpha on hepatitis B virus following lamivudine treatment. World J. Gastroenterol., 2012, vol. 18, no. 27, pp. 3617–3622. doi: 10.3748/wjg.v18.i27.3617
- Uusi-Mäkelä M.I.E., Barker H.R., Bäuerlein C.A., Häkkinen T., Nykter M., Rämet M. Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio). PLoS One, 2018, vol. 13, no. 4: e0196238–e0196238. doi: 10.1371/journal. pone.0196238
- Verkuijl S.A., Rots M.G. The influence of eukaryotic chromatin state on CRISPR-Cas9 editing efficiencies. Curr. Opin. Biotechnol., 2018, vol. 55, pp. 68–73. doi: 10.1016/j.copbio.2018.07.005
- WHO. Global hepatitis report 2017. World Health Organization, 2017.
- Yue D., Zhang Y., Cheng L., Ma J., Xi Y., Yang L., Xiang R. Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by 1 H-NMR-based metabonomics. Sci. Rep., 2016, vol. 6: 24430. doi: 10.1038/srep24430
Supplementary files
СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).