Modern Approaches to Molecular Genetics of Viruses in the Study of the Members of the Family Coronaviridae

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.

1. Yount 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.74.22.10600-10611.2000

2. Yount 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.76.21.11065-11078.2002

3. Ren 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.01085-07

4. Thao 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/s41586-020-2294-9

5. Xie 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.2020.04.004

6. Xie 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/s41596-021-00491-8

7. Ye 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.02168-20

8. Rihn 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.3001091

9. 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/)

10. 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/10.35825/2587-5728-2020-4-4-421-430

11. Lakota 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/10.35825/2587-5728-2021-5-2-103-122

12. Godeke 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.74.3.1566-1571.2000

13. Opstelten 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.131.2.339

14. Kuo 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.74.3.1393-1406.2000

15. Perez 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_5

16. Yount 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.1735582100

17. Kouprina 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.2008.5

18. de 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.79.22.14451-14456.2005

19. Follis 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.2006.02.003

20. Eckert 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.70.1.777

21. Watanabe 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.01412-08

22. Rockx 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.00505-07

23. Becker 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.0808116105

24. Sheahan 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.02041-07

25. Bolles 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.06048-11

26. Li F. Structure, function, and evolution of coronavirus spike proteins // Ann. Rev. Virol. 2016. V. 29. P. 237–261. https://doi.org/10.1146/annurevvirology-110615-042301

27. Menachery 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.1517719113

28. 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.

29. 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.1009840

30. Medical aspects of chemical and biological warfare / Ed. Sidell F.R., Tafuqi E.T., Franz D.R. Washington. 1997.

31. 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.75.3.1205-1210.2001

32. Cheng 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/v11100972

33. Nagy 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.2020.106272

34. Segreto 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/s10311-021-01211-0

35. Markson S. What really happened in Wuhan. Harper Collins Publishers Australia Pty Limited. ISBN 978 1 4607 6092 5. 2021.