nbsprotВестник войск РХБ защитыJournal of NBC Protection Corps2587-57283034-279127 Научный центр10.35825/2587-5728-2021-1-3-217-235vwsibbnbsprot-67Research ArticleБиологическая безопасность и защита от биологических угрозBiological Security and Protection against Biological ThreatsСовременные подходы в области молекулярной генетики вирусов при изучении представителей семейства CoronaviridaeModern Approaches to Molecular Genetics of Viruses in the Study of the Members of the Family CoronaviridaeСупотницкийМ. В.SupotnitskiyM. V.

Супотницкий Михаил Васильевич. Главный специалист, канд. биол. наук, ст. науч. сотр.

105005, Москва, Бригадирский переулок, д. 13

Mikhail Vasilyevich Supotnitskiy. Senior Researcher. Chief Specialist. Candidate of Biological Sciences.

Brigadirskii Lane 13, Moscow 105005

27nc_1@mil.ruФедеральное государственное бюджетное учреждение «27 Научный центр» Министерства обороны Российской ФедерацииРоссияFederal State Budgetary Establishment «27 Scientific Centre» of the Ministry of Defence of the Russian FederationRussian Federation20211506202153217235Copyright © Супотницкий М.В., 20212021Супотницкий М.В.Supotnitskiy M.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.nbsprot.ru/jour/article/view/67

Существующие подозрения об искусственном происхождении пандемии COVID-19 и об использовании технологии обратной генетики для создания вируса SARS-CoV-2 требуют понимания ее возможностей в конструировании новых вирусов. Цель работы – показать, каким образом применение обратной генетики позволяет конструировать ранее не существовавшие коронавирусы, технологии и основные достижения в их создании. Для подготовки данной статьи использовалась информация, находящаяся в открытом доступе и легко проверяемая по приведенным источникам. Название технологии – «обратная генетика» – произошло из-за того, что при получении способных к размножению РНК-вирусов идут не от ДНК к РНК, как это обычно делается в клетке при синтезе белка, а наоборот, от РНК вируса к комплементарной ей ДНК (кДНК), а с нее с помощью РНК-полимеразы фага Т7 – «обратно» к инфекционной РНК. Так как полученная плюс-РНК генома коронавируса имитирует клеточную матричную РНК (мРНК), она немедленно распознается машиной трансляции клетки и запускает формирование собственных инфекционных вирусных частиц. Разработано две системы обратной генетики, предполагающие получение инфекционной плюс-РНК – в условиях in vitro и in vivo. Проблема получения полноразмерной кДНК гигантского генома коронавирусов решается путем его фрагментации и последующей сшивки фрагментов с использованием стандартных подходов молекулярной биологии. В статье приведены примеры, каким образом данная технология позволяет получать синтетические коронавирусы, по свойствам неотличимые от выделенных из природы, менять круг их хозяев, усиливать вирулентность и устойчивость к специфическим антителам, влиять на патогенез болезни. Также показаны перспективы использования рекомбинантных вирусов в клеточных скрининговых анализах и моделях инфекции in vivo для идентификации профилактических и терапевтических подходов к лечению вирусных инфекций.

The existence of certain suspicions about the artificial origin of the COVID-19 pandemic and about the possible use of reverse genetics technology to create the SARS-CoV-2 virus require an understanding of its capabilities in the design of new viruses. The aim of this work is to show how the use of reverse genetics allows the design of previously non-existent coronaviruses, technologies and the main achievements in their creation. Only the information in the public domain was used for the preparation of this article. The technology is called «reverse genetics» because when obtaining RNA viruses capable of replication, the process is going not from DNA to RNA, as usual, but on the contrary, from the RNA of the virus to its complementary DNA (cDNA), and from it with the help of T7 RNA polymerase – «back» to the infectious RNA. Since the resulting plus-RNA of the coronavirus genome mimics cellular messenger RNA (mRNA), it is immediately recognized by the cell's translation machine and triggers the formation of its own infectious viral particles. Two systems of reverse genetics have been developed, involving the production of an infectious plusRNA, in vitro and in vivo. The problem of obtaining a full-length cDNA of the giant genome of coronaviruses is solved by fragmentation and subsequent stitching of fragments using standard molecular biology approaches. The article provides the examples of how this technology makes it possible to obtain synthetic coronaviruses that are indistinguishable from those isolated from nature, to change the range of their hosts, to enhance virulence and resistance to specific antibodies, and to influence the pathogenesis of the disease. The article also shows the prospects for the use of recombinant viruses in cellular screening analyses and infection models in vivo for the identification of preventive and therapeutic approaches to the virus disease treatment.

атипичная пневмониякоронавирусмежвидовая передачаобратная генетикапандемияCOVID-19SARS-CoV-2atypical pneumoniaсoronavirusinterspecies transmissionreserve geneticspandemicCOVID-19SARS-CoV-2ReferencesYount B., Curtis K., Baric R. Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model // J. Virol. 2000. V. 74(22). P. 10600–10611. https://doi.org/10.1128/JVI.74.22.10600-10611.2000Yount B., Curtis K., Baric R. Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model // J. Virol. 2000. V. 74(22). P. 10600–10611. https://doi.org/10.1128/JVI.74.22.10600-10611.2000Yount B., Denison M. Weiss S., Baric R. Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59 // J. Virol. 2002. V. 76(21). P. 11065–11078. https://doi.org/10.1128/JVI.76.21.11065-11078.2002Yount B., Denison M. Weiss S., Baric R. Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59 // J. Virol. 2002. V. 76(21). P. 11065–11078. https://doi.org/10.1128/JVI.76.21.11065-11078.2002Ren W., QuX., Li W., Shi1 Z. et al. Difference in receptor usage between Severe Acute Respiratory Syndrome (SARS) coronavirus and SARS-Like coronavirus of bat origin // J. Virol. 2008. V. 82(4). P. 1899–1907. https://doi.org/10.1128/JVI.01085-07Ren W., QuX., Li W., Shi1 Z. et al. Difference in receptor usage between Severe Acute Respiratory Syndrome (SARS) coronavirus and SARS-Like coronavirus of bat origin // J. Virol. 2008. V. 82(4). P. 1899–1907. https://doi.org/10.1128/JVI.01085-07Thao T.T.N., Labroussaa F., Thiel V. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform // Nature. 2020. V. 582. P. 561–565. https://doi.org/10.1038/s41586-020-2294-9Thao T.T.N., Labroussaa F., Thiel V. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform // Nature. 2020. V. 582. P. 561–565. https://doi.org/10.1038/s41586-020-2294-9Xie X., Muruato A., Lokugamage K.G. et al. An infectious cDNA clone of SARS-CoV-2 // Cell Host Microbe. 2020 May 13. V. 27(5). P. 841–848.e3. https://doi.org/10.1016/j.chom.2020.04.004Xie X., Muruato A., Lokugamage K.G. et al. An infectious cDNA clone of SARS-CoV-2 // Cell Host Microbe. 2020 May 13. V. 27(5). P. 841–848.e3. https://doi.org/10.1016/j.chom.2020.04.004Xie X., Lokugamage K.G., Zhang X. et al. Engineering SARS-CoV-2 using a reverse genetic system // Nat. Protoc. 2021. V. 16(3). P. 1761–1784. https://doi.org/10.1038/s41596-021-00491-8Xie X., Lokugamage K.G., Zhang X. et al. Engineering SARS-CoV-2 using a reverse genetic system // Nat. Protoc. 2021. V. 16(3). P. 1761–1784. https://doi.org/10.1038/s41596-021-00491-8Ye Ch., Chiem K., Park J-G et al. Rescue of SARSCoV-2 from a single bacterial artificial chromosome // mBio. 2020. 11(5): e02168-20. https://doi.org/10.1128/mBio.02168-20Ye Ch., Chiem K., Park J-G et al. Rescue of SARSCoV-2 from a single bacterial artificial chromosome // mBio. 2020. 11(5): e02168-20. https://doi.org/10.1128/mBio.02168-20Rihn S., Merits A., Bakshi S. et al. A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research // PLoS Biol. 2021. V. 19(2). e3001091. https://doi.org/10.1371/journal.pbio.3001091Rihn S., Merits A., Bakshi S. et al. A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research // PLoS Biol. 2021. V. 19(2). e3001091. https://doi.org/10.1371/journal.pbio.3001091Menachery V.D., Yount B.L., Debbink K. et al. SARS-like cluster of circulating bat coronavirus pose threat for human emergence // Nat. Med. 2015. V. 21, № 12. P. 1508–1513. https://doi.org/10.1038/nm.3985/(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797993/)Menachery V.D., Yount B.L., Debbink K. et al. SARS-like cluster of circulating bat coronavirus pose threat for human emergence // Nat. Med. 2015. V. 21, № 12. P. 1508–1513. https://doi.org/10.1038/nm.3985/(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797993/)Lakota J. Today`s Biothreats – Where the Past Predictions Meet the Future // Journal of NBC Protection Corps. 2020. V. 4. No 4. P. 421–440. https://doi.org/10.35825/2587-5728-2020-4-4-421-430Lakota J. Today`s Biothreats – Where the Past Predictions Meet the Future // Journal of NBC Protection Corps. 2020. V. 4. No 4. P. 421–440. https://doi.org/10.35825/2587-5728-2020-4-4-421-430Lakota J. Synthetic Biology – Friend or Foe? What Kind of Threats Should We Expect? // Journal of NBC Protection Corps. 2021. V. 5. № 2. P. 103–122. https://doi.org/10.35825/2587-5728-2021-5-2-103-122Lakota J. Synthetic Biology – Friend or Foe? What Kind of Threats Should We Expect? // Journal of NBC Protection Corps. 2021. V. 5. № 2. P. 103–122. https://doi.org/10.35825/2587-5728-2021-5-2-103-122Godeke G-J., de Haan C.A.M., Rossen J.W.A. et al. Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein // J. Virol. 1999. V. 74. P. 1566–1571. https://doi.org/10.1128/jvi.74.3.1566-1571.2000Godeke G-J., de Haan C.A.M., Rossen J.W.A. et al. Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein // J. Virol. 1999. V. 74. P. 1566–1571. https://doi.org/10.1128/jvi.74.3.1566-1571.2000Opstelten D-J.E., Raamsman M.J.B, Wolfs K. et al. Envelope glycoprotein interactions in coronavirus assembly // J. Cell Biol. 1995. V. 131. P. 339–349. https://doi.org/10.1083/jcb.131.2.339Opstelten D-J.E., Raamsman M.J.B, Wolfs K. et al. Envelope glycoprotein interactions in coronavirus assembly // J. Cell Biol. 1995. V. 131. P. 339–349. https://doi.org/10.1083/jcb.131.2.339Kuo L., Godeke G-J., Raamsman M.J. et al. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier // J. Virol. 2000. V. 74(3). P. 1393–1406. https://doi.org/10.1128/jvi.74.3.1393-1406.2000Kuo L., Godeke G-J., Raamsman M.J. et al. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier // J. Virol. 2000. V. 74(3). P. 1393–1406. https://doi.org/10.1128/jvi.74.3.1393-1406.2000Perez D.R., Cockrell A.S., Beall A. et al. Efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses // Reverse Genetics of RNA Viruses. 2017. V. 1602. P. 59– 81. https://doi.org/10.1007/978-1-4939-6964-7_5Perez D.R., Cockrell A.S., Beall A. et al. Efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses // Reverse Genetics of RNA Viruses. 2017. V. 1602. P. 59– 81. https://doi.org/10.1007/978-1-4939-6964-7_5Yount B., Curtis K.M., Fritz E.A. et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus // Proc. Natl. Acad. Sci. USA. 2003. V. 100(22). P. 12995–13000. https://doi.org/10.1073/pnas.1735582100Yount B., Curtis K.M., Fritz E.A. et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus // Proc. Natl. Acad. Sci. USA. 2003. V. 100(22). P. 12995–13000. https://doi.org/10.1073/pnas.1735582100Kouprina N., Larionov V. Selective isolation of genomic loci from complex genomes by transformationassociated recombination cloning in the yeast Saccharomyces cerevisiae // Nat. Protoc. 2008. V. 3(3). P. 371–377. https://doi.org/10.1038/nprot.2008.5Kouprina N., Larionov V. Selective isolation of genomic loci from complex genomes by transformationassociated recombination cloning in the yeast Saccharomyces cerevisiae // Nat. Protoc. 2008. V. 3(3). P. 371–377. https://doi.org/10.1038/nprot.2008.5de Haan С., Li Z., Lintelo E. et al. Murine coronavirus with an extended host range uses heparan sulfate as an entry receptor // J. Virol. 2005. V. 79(22). P. 14451–14456. https://doi.org/10.1128/JVI.79.22.14451-14456.2005de Haan С., Li Z., Lintelo E. et al. Murine coronavirus with an extended host range uses heparan sulfate as an entry receptor // J. Virol. 2005. V. 79(22). P. 14451–14456. https://doi.org/10.1128/JVI.79.22.14451-14456.2005Follis K.E., York J., Nunberg J.H. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry // Virology. 2006. V. 350(2). P. 358–369. https://doi.org/10.1016/j.virol.2006.02.003Follis K.E., York J., Nunberg J.H. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry // Virology. 2006. V. 350(2). P. 358–369. https://doi.org/10.1016/j.virol.2006.02.003Eckert D.M., Kim P.S. Mechanisms of viral membrane fusion and its inhibition // Ann.l Rev. Biochem. 2001. V. 70. P. 777–810. https://doi.org/10.1146/annurev.biochem.70.1.777Eckert D.M., Kim P.S. Mechanisms of viral membrane fusion and its inhibition // Ann.l Rev. Biochem. 2001. V. 70. P. 777–810. https://doi.org/10.1146/annurev.biochem.70.1.777Watanabe R., Matsuyama S., Shirato K. et al. Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein // J. Virol. 2008. V. 82(23). P. 11985–11991. https://doi.org/10.1128/JVI.01412-08Watanabe R., Matsuyama S., Shirato K. et al. Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein // J. Virol. 2008. V. 82(23). P. 11985–11991. https://doi.org/10.1128/JVI.01412-08Rockx B., Sheahan T., Donaldson E. et al. Synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice // J. Virol. 2007. V. 81. P. 7410–7423. https://doi.org/10.1128/JVI.00505-07Rockx B., Sheahan T., Donaldson E. et al. Synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice // J. Virol. 2007. V. 81. P. 7410–7423. https://doi.org/10.1128/JVI.00505-07Becker M.M., Graham R.L., Donaldson F. et al. Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice // Proc. Natl. Acad. Sci. USA. 2008. V. 105(50). P. 19944–19949. https://doi.org/10.1073/pnas.0808116105Becker M.M., Graham R.L., Donaldson F. et al. Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice // Proc. Natl. Acad. Sci. USA. 2008. V. 105(50). P. 19944–19949. https://doi.org/10.1073/pnas.0808116105Sheahan T. Rockx B., Donaldson E. et al. Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium // J. Virol. 2008. V. 82. P. 2274–2285. https://doi.org/10.1128/JVI.02041-07Sheahan T. Rockx B., Donaldson E. et al. Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium // J. Virol. 2008. V. 82. P. 2274–2285. https://doi.org/10.1128/JVI.02041-07Bolles M., Deming D., Long K. et al. A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge // J. Virol. 2011. V. 85. P. 12201–12215. https://doi.org/10.1128/JVI.06048-11Bolles M., Deming D., Long K. et al. A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge // J. Virol. 2011. V. 85. P. 12201–12215. https://doi.org/10.1128/JVI.06048-11Li F. Structure, function, and evolution of coronavirus spike proteins // Ann. Rev. Virol. 2016. V. 29. P. 237–261. https://doi.org/10.1146/annurevvirology-110615-042301Li F. Structure, function, and evolution of coronavirus spike proteins // Ann. Rev. Virol. 2016. V. 29. P. 237–261. https://doi.org/10.1146/annurevvirology-110615-042301Menachery V., Yount B., Sims A. et al. SARSlike WIV1-CoV poised for human emergence // PNAS. 2016. V. 113 (11). P. 3048–3053. https://doi.org/10.1073/pnas.1517719113Menachery V., Yount B., Sims A. et al. SARSlike WIV1-CoV poised for human emergence // PNAS. 2016. V. 113 (11). P. 3048–3053. https://doi.org/10.1073/pnas.1517719113Sia S.F., Yan L-M., Chin A.W.H. et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters // Nature. 2020. V. 583. P. 834–838. https://doi.org/10.1038/s41586-020-2342-5.Sia S.F., Yan L-M., Chin A.W.H. et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters // Nature. 2020. V. 583. P. 834–838. https://doi.org/10.1038/s41586-020-2342-5.Pickard A., Calverley B., Chang J. et al. Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells // PLoS Pathog. 2021. V. 17(9). e1009840. https://doi.org/10.1371/journal.ppat.1009840Pickard A., Calverley B., Chang J. et al. Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells // PLoS Pathog. 2021. V. 17(9). e1009840. https://doi.org/10.1371/journal.ppat.1009840Medical aspects of chemical and biological warfare / Ed. Sidell F.R., Tafuqi E.T., Franz D.R. Washington. 1997.Medical aspects of chemical and biological warfare / Ed. Sidell F.R., Tafuqi E.T., Franz D.R. Washington. 1997.Jackson P.J., Ramsay A.J., Christensen C.D. et al. Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistаnce to mousepox // J. Virol. 2001. V. 75(3). P. 1205–1210. https://doi.org/10.1128/JVI.75.3.1205-1210.2001Jackson P.J., Ramsay A.J., Christensen C.D. et al. Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistаnce to mousepox // J. Virol. 2001. V. 75(3). P. 1205–1210. https://doi.org/10.1128/JVI.75.3.1205-1210.2001Cheng J., Zhao Y., Xu G. et al. The S2 subunit of QX-type infectious bronchitis coronavirus spike protein is an essential determinant of neurotropism // Viruses. 2019. V. 11(10). 972. https://doi.org/10.3390/v11100972Cheng J., Zhao Y., Xu G. et al. The S2 subunit of QX-type infectious bronchitis coronavirus spike protein is an essential determinant of neurotropism // Viruses. 2019. V. 11(10). 972. https://doi.org/10.3390/v11100972Nagy A., Pongor S., Gyorffy B. Different mutations in SARS-CoV-2 associate with severe and mild outcome // Int. J. Antimicrob. Agents. 2021. V. 57. P. 106272. https://doi.org/10.1016/j.ijantimicag.2020.106272Nagy A., Pongor S., Gyorffy B. Different mutations in SARS-CoV-2 associate with severe and mild outcome // Int. J. Antimicrob. Agents. 2021. V. 57. P. 106272. https://doi.org/10.1016/j.ijantimicag.2020.106272Segreto R., Deigin Y., McCairn K., Sousa A. et al. Should we discount the laboratory origin of COVID-19? // Environ. Chem. Lett. 2021. Mar 25. P. 1–15. https://doi.org/10.1007/s10311-021-01211-0Segreto R., Deigin Y., McCairn K., Sousa A. et al. Should we discount the laboratory origin of COVID-19? // Environ. Chem. Lett. 2021. Mar 25. P. 1–15. https://doi.org/10.1007/s10311-021-01211-0Markson S. What really happened in Wuhan. Harper Collins Publishers Australia Pty Limited. ISBN 978 1 4607 6092 5. 2021.Markson S. What really happened in Wuhan. Harper Collins Publishers Australia Pty Limited. ISBN 978 1 4607 6092 5. 2021.

The authors declare that there are no conflicts of interest present.