Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins

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 analo...

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Autores principales:	S. A. Brezgin, A. P. Kostyusheva, V. N. Simirsky, E. V. Volchkova, D. S. Chistyakov, D. S. Kostyushev, V. P. Chulanov
Formato:	article
Lenguaje:	RU
Publicado:	Sankt-Peterburg : NIIÈM imeni Pastera 2019
Materias:	hepatitis b virus circular covalently closed dna crispr/cas9 specificity hbx protein mutant forms of hbx protein Infectious and parasitic diseases RC109-216
Acceso en línea:	https://doaj.org/article/46e337894222405ebf01cb8c6a0c49f4
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spelling	oai:doaj.org-article:46e337894222405ebf01cb8c6a0c49f42021-11-22T07:09:53ZSuppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins2220-76192313-739810.15789/2220-7619-2019-3-4-476-484https://doaj.org/article/46e337894222405ebf01cb8c6a0c49f42019-11-01T00:00:00Zhttps://www.iimmun.ru/iimm/article/view/922https://doaj.org/toc/2220-7619https://doaj.org/toc/2313-7398Chronic 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.S. A. BrezginA. P. KostyushevaV. N. SimirskyE. V. VolchkovaD. S. ChistyakovD. S. KostyushevV. P. ChulanovSankt-Peterburg : NIIÈM imeni Pasteraarticlehepatitis b viruscircular covalently closed dnacrispr/cas9specificityhbx proteinmutant forms of hbx proteinInfectious and parasitic diseasesRC109-216RUInfekciâ i Immunitet, Vol 9, Iss 3-4, Pp 476-484 (2019)
institution	DOAJ
collection	DOAJ
language	RU
topic	hepatitis b virus circular covalently closed dna crispr/cas9 specificity hbx protein mutant forms of hbx protein Infectious and parasitic diseases RC109-216
spellingShingle	hepatitis b virus circular covalently closed dna crispr/cas9 specificity hbx protein mutant forms of hbx protein Infectious and parasitic diseases RC109-216 S. A. Brezgin A. P. Kostyusheva V. N. Simirsky E. V. Volchkova D. S. Chistyakov D. S. Kostyushev V. P. Chulanov Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
description	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.
format	article
author	S. A. Brezgin A. P. Kostyusheva V. N. Simirsky E. V. Volchkova D. S. Chistyakov D. S. Kostyushev V. P. Chulanov
author_facet	S. A. Brezgin A. P. Kostyusheva V. N. Simirsky E. V. Volchkova D. S. Chistyakov D. S. Kostyushev V. P. Chulanov
author_sort	S. A. Brezgin
title	Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
title_short	Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
title_full	Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
title_fullStr	Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
title_full_unstemmed	Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins
title_sort	suppression of hepatitis b virus by a combined activity of crispr/cas9 and hbx proteins
publisher	Sankt-Peterburg : NIIÈM imeni Pastera
publishDate	2019
url	https://doaj.org/article/46e337894222405ebf01cb8c6a0c49f4
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Suppression of hepatitis b virus by a combined activity of CRISPR/Cas9 and HBx proteins

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