Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level SARS-CoV-2 variant changes protein and RNA level

Main Article Content

Shay Leary
Silvana Gaudieri
Matthew D. Parker
Abha Chopra
Ian James
Suman Pakala
Eric Alves
Mina John
Benjamin B. Lindsey
Alexander J. Keeley
Sarah L. Rowland-Jones
Maurice S. Swanson
David A. Ostrov
Jodi L. Bubenik
Suman R. Das
John Sidney
Alessandro Sette
COVID-19 Genomics UK (COG-UK) consortium
Thushan I. de Silva
Elizabeth Phillips
Simon Mallal

Abstract

Background: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. 


Methods: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. 


Conclusions: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

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References

Zhang T, Wu Q, Zhang Z. Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak. Curr Biol. 2020;30(7):1346-51 e2. doi: 10.1016/j.cub.2020.03.022. PubMed PMID: 32197085; PMCID: PMC7156161.

Korber B, Fischer WM, Gnanakaran S, Yoon H, Theiler J, Abfalterer W, Hengartner N, Giorgi EE, Bhattacharya T, Foley B, Hastie KM, Parker MD, Partridge DG, Evans CM, Freeman TM, de Silva TI, Sheffield C-GG, McDanal C, Perez LG, Tang H, Moon-Walker A, Whelan SP, LaBranche CC, Saphire EO, Montefiori DC. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell. 2020;182(4):812-27 e19. doi: 10.1016/j.cell.2020.06.043. PubMed PMID: 32697968; PMCID: PMC7332439.

Grubaugh ND, Hanage WP, Rasmussen AL. Making Sense of Mutation: What D614G Means for the COVID-19 Pandemic Remains Unclear. Cell. 2020;182(4):794-5. doi: 10.1016/j.cell.2020.06.040. PubMed PMID: 32697970; PMCID: PMC7332445.

Yurkovetskiy L, Wang X, Pascal KE, Tomkins-Tinch C, Nyalile T, Wang Y, Baum A, Diehl WE, Dauphin A, Carbone C, Veinotte K, Egri SB, Schaffner SF, Lemieux JE, Munro J, Rafique A, Barve A, Sabeti PC, Kyratsous CA, Dudkina N, Shen K, Luban J. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. bioRxiv. 2020. doi: 10.1101/2020.07.04.187757. PubMed PMID: 32637944; PMCID: PMC7337374.

Zhang L, Jackson CB, Mou H, Ojha A, Rangarajan ES, Izard T, Farzan M, Choe H. The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity. bioRxiv. 2020. doi: 10.1101/2020.06.12.148726. PubMed PMID: 32587973; PMCID: PMC7310631.

Graham RL, Baric RS. Recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission. J Virol. 2010;84(7):3134-46. doi: 10.1128/JVI.01394-09. PubMed PMID: 19906932; PMCID: PMC2838128.

Ji W, Wang W, Zhao X, Zai J, Li X. Cross-species transmission of the newly identified coronavirus 2019-nCoV. J Med Virol. 2020;92(4):433-40. doi: 10.1002/jmv.25682. PubMed PMID: 31967321; PMCID: PMC7138088.

Hertzman RJ, Deshpande P, Leary S, Li Y, Ram R, Chopra A, Cooper D, Watson M, Palubinsky AM, Mallal S, Gibson A, Phillips EJ. Visual Genomics Analysis Studio as a Tool to Analyze Multiomic Data. Front Genet. 2021;12:642012. doi: 10.3389/fgene.2021.642012. PubMed PMID: 34220932; PMCID: PMC8247644.

Sidney J, Southwood S, Moore C, Oseroff C, Pinilla C, Grey HM, Sette A. Measurement of MHC/peptide interactions by gel filtration or monoclonal antibody capture. Curr Protoc Immunol. 2013;Chapter 18:Unit 18 3. doi: 10.1002/0471142735.im1803s100. PubMed PMID: 23392640; PMCID: PMC3626435.

Sidney J, del Guercio MF, Southwood S, Engelhard VH, Appella E, Rammensee HG, Falk K, Rotzschke O, Takiguchi M, Kubo RT, et al. Several HLA alleles share overlapping peptide specificities. J Immunol. 1995;154(1):247-59. PubMed PMID: 7527812.

Parker MD, Lindsey BB, Leary S, Gaudieri S, Chopra A, Wyles M, Angyal A, Green LR, Parsons P, Tucker RM, Brown R, Groves D, Johnson K, Carrilero L, Heffer J, Partridge DG, Evans C, Raza M, Keeley AJ, Smith N, Filipe ADS, Shepherd JG, Davis C, Bennett S, Sreenu VB, Kohl A, Aranday-Cortes E, Tong L, Nichols J, Thomson EC, Consortium C-GU, Wang D, Mallal S, de Silva TI. Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data. Genome Res. 2021;31(4):645-58. doi: 10.1101/gr.268110.120. PubMed PMID: 33722935; PMCID: PMC8015849.

Jenjaroenpun P, Wanchai V, Ono-Moore KD, Laudadio J, James LP, Adams SH, Prior F, Nookaew I, Ussery DW, Wongsurawat T. Two SARS-CoV-2 Genome Sequences of Isolates from Rural U.S. Patients Harboring the D614G Mutation, Obtained Using Nanopore Sequencing. Microbiol Resour Announc. 2020;10(1). doi: 10.1128/MRA.01109-20. PubMed PMID: 33334896.

Franco-Munoz C, Alvarez-Diaz DA, Laiton-Donato K, Wiesner M, Escandon P, Usme-Ciro JA, Franco-Sierra ND, Florez-Sanchez AC, Gomez-Rangel S, Rodriguez-Calderon LD, Barbosa-Ramirez J, Ospitia-Baez E, Walteros DM, Ospina-Martinez ML, Mercado-Reyes M. Substitutions in Spike and Nucleocapsid proteins of SARS-CoV-2 circulating in South America. Infect Genet Evol. 2020;85:104557. doi: 10.1016/j.meegid.2020.104557. PubMed PMID: 32950697; PMCID: PMC7497549.

Leslie A, Kavanagh D, Honeyborne I, Pfafferott K, Edwards C, Pillay T, Hilton L, Thobakgale C, Ramduth D, Draenert R, Le Gall S, Luzzi G, Edwards A, Brander C, Sewell AK, Moore S, Mullins J, Moore C, Mallal S, Bhardwaj N, Yusim K, Phillips R, Klenerman P, Korber B, Kiepiela P, Walker B, Goulder P. Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA. J Exp Med. 2005;201(6):891-902. doi: 10.1084/jem.20041455. PubMed PMID: 15781581; PMCID: PMC2213090.

Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, Feeney M, Tang Y, Holmes EC, Allen T, Prado JG, Altfeld M, Brander C, Dixon C, Ramduth D, Jeena P, Thomas SA, St John A, Roach TA, Kupfer B, Luzzi G, Edwards A, Taylor G, Lyall H, Tudor-Williams G, Novelli V, Martinez-Picado J, Kiepiela P, Walker BD, Goulder PJ. HIV evolution: CTL escape mutation and reversion after transmission. Nat Med. 2004;10(3):282-9. doi: 10.1038/nm992. PubMed PMID: 14770175.

Moore CB, John M, James IR, Christiansen FT, Witt CS, Mallal SA. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science. 2002;296(5572):1439-43. doi: 10.1126/science.1069660. PubMed PMID: 12029127.

Fitzmaurice K, Petrovic D, Ramamurthy N, Simmons R, Merani S, Gaudieri S, Sims S, Dempsey E, Freitas E, Lea S, McKiernan S, Norris S, Long A, Kelleher D, Klenerman P. Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection. Gut. 2011;60(11):1563-71. doi: 10.1136/gut.2010.228403. PubMed PMID: 21551190; PMCID: PMC3184218.

Rubnitz J, Subramani S. The minimum amount of homology required for homologous recombination in mammalian cells. Mol Cell Biol. 1984;4(11):2253-8. doi: 10.1128/mcb.4.11.2253-2258.1984. PubMed PMID: 6096689; PMCID: PMC369052.

Sola I, Moreno JL, Zuniga S, Alonso S, Enjuanes L. Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis. J Virol. 2005;79(4):2506-16. doi: 10.1128/JVI.79.4.2506-2516.2005. PubMed PMID: 15681451; PMCID: PMC546574.

Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The Architecture of SARS-CoV-2 Transcriptome. Cell. 2020;181(4):914-21 e10. doi: 10.1016/j.cell.2020.04.011. PubMed PMID: 32330414; PMCID: PMC7179501.

Chandrashekar A, Liu J, Martinot AJ, McMahan K, Mercado NB, Peter L, Tostanoski LH, Yu J, Maliga Z, Nekorchuk M, Busman-Sahay K, Terry M, Wrijil LM, Ducat S, Martinez DR, Atyeo C, Fischinger S, Burke JS, Slein MD, Pessaint L, Van Ry A, Greenhouse J, Taylor T, Blade K, Cook A, Finneyfrock B, Brown R, Teow E, Velasco J, Zahn R, Wegmann F, Abbink P, Bondzie EA, Dagotto G, Gebre MS, He X, Jacob-Dolan C, Kordana N, Li Z, Lifton MA, Mahrokhian SH, Maxfield LF, Nityanandam R, Nkolola JP, Schmidt AG, Miller AD, Baric RS, Alter G, Sorger PK, Estes JD, Andersen H, Lewis MG, Barouch DH. SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science. 2020;369(6505):812-7. doi: 10.1126/science.abc4776. PubMed PMID: 32434946; PMCID: PMC7243369.

Wolfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Muller MA, Niemeyer D, Jones TC, Vollmar P, Rothe C, Hoelscher M, Bleicker T, Brunink S, Schneider J, Ehmann R, Zwirglmaier K, Drosten C, Wendtner C. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020;581(7809):465-9. doi: 10.1038/s41586-020-2196-x. PubMed PMID: 32235945.

Lorenz R, Bernhart SH, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL. ViennaRNA Package 2.0. Algorithms Mol Biol. 2011;6:26. doi: 10.1186/1748-7188-6-26. PubMed PMID: 22115189; PMCID: PMC3319429.

Laing C, Wen D, Wang JT, Schlick T. Predicting coaxial helical stacking in RNA junctions. Nucleic Acids Res. 2012;40(2):487-98. doi: 10.1093/nar/gkr629. PubMed PMID: 21917853; PMCID: PMC3258123.

de la Pena M, Dufour D, Gallego J. Three-way RNA junctions with remote tertiary contacts: a recurrent and highly versatile fold. RNA. 2009;15(11):1949-64. doi: 10.1261/rna.1889509. PubMed PMID: 19741022; PMCID: PMC2764472.

Hua L, Song Y, Kim N, Laing C, Wang JT, Schlick T. CHSalign: A Web Server That Builds upon Junction-Explorer and RNAJAG for Pairwise Alignment of RNA Secondary Structures with Coaxial Helical Stacking. PloS One. 2016;11(1):e0147097. doi: 10.1371/journal.pone.0147097. PubMed PMID: 26789998; PMCID: PMC4720362.

Chapman EG, Moon SL, Wilusz J, Kieft JS. RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA. eLife. 2014;3:e01892. doi: 10.7554/eLife.01892. PubMed PMID: 24692447; PMCID: PMC3968743.

Gaudieri S, Rauch A, Park LP, Freitas E, Herrmann S, Jeffrey G, Cheng W, Pfafferott K, Naidoo K, Chapman R, Battegay M, Weber R, Telenti A, Furrer H, James I, Lucas M, Mallal SA. Evidence of viral adaptation to HLA class I-restricted immune pressure in chronic hepatitis C virus infection. J Virol. 2006;80(22):11094-104. doi: 10.1128/JVI.00912-06. PubMed PMID: 17071929; PMCID: PMC1642167.

Brumme ZL, Kinloch NN, Sanche S, Wong A, Martin E, Cobarrubias KD, Sandstrom P, Levett PN, Harrigan PR, Joy JB. Extensive host immune adaptation in a concentrated North American HIV epidemic. Aids. 2018;32(14):1927-38. doi: 10.1097/QAD.0000000000001912. PubMed PMID: 30048246; PMCID: PMC6125742.

Katoh J, Kawana-Tachikawa A, Shimizu A, Zhu D, Han C, Nakamura H, Koga M, Kikuchi T, Adachi E, Koibuchi T, Gao GF, Brumme ZL, Iwamoto A. Rapid HIV-1 Disease Progression in Individuals Infected with a Virus Adapted to Its Host Population. PloS One. 2016;11(3):e0150397. doi: 10.1371/journal.pone.0150397. PubMed PMID: 26953793; PMCID: PMC4783116.

Peng H, Yang LT, Wang LY, Li J, Huang J, Lu ZQ, Koup RA, Bailer RT, Wu CY. Long-lived memory T lymphocyte responses against SARS coronavirus nucleocapsid protein in SARS-recovered patients. Virology. 2006;351(2):466-75. doi: 10.1016/j.virol.2006.03.036. PubMed PMID: 16690096; PMCID: PMC7111820.

Yang Y, Yan W, Hall B, Jiang X. Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination. bioRxiv. 2020. doi: 10.1101/2020.06.21.163410. PubMed PMID: 32587968; PMCID: PMC7310624.

Sola I, Almazan F, Zuniga S, Enjuanes L. Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annu Rev Virol. 2015;2(1):265-88. doi: 10.1146/annurev-virology-100114-055218. PubMed PMID: 26958916; PMCID: PMC6025776.

Kopecky-Bromberg SA, Martinez-Sobrido L, Frieman M, Baric RA, Palese P. Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists. J Virol. 2007;81(2):548-57. doi: 10.1128/JVI.01782-06. PubMed PMID: 17108024; PMCID: PMC1797484.

Lokugamage KG, Hage A, de Vries M, Valero-Jimenez AM, Schindewolf C, Dittmann M, Rajsbaum R, Menachery VD. Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV. bioRxiv. 2020. doi: 10.1101/2020.03.07.982264. PubMed PMID: 32511335; PMCID: PMC7239075.

Hu Y, Li W, Gao T, Cui Y, Jin Y, Li P, Ma Q, Liu X, Cao C. The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination. J Virol. 2017;91(8). doi: 10.1128/JVI.02143-16. PubMed PMID: 28148787; PMCID: PMC5375661.

Manokaran G, Finol E, Wang C, Gunaratne J, Bahl J, Ong EZ, Tan HC, Sessions OM, Ward AM, Gubler DJ, Harris E, Garcia-Blanco MA, Ooi EE. Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness. Science. 2015;350(6257):217-21. doi: 10.1126/science.aab3369. PubMed PMID: 26138103; PMCID: PMC4824004.

Zuniga S, Cruz JL, Sola I, Mateos-Gomez PA, Palacio L, Enjuanes L. Coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription. J Virol. 2010;84(4):2169-75. doi: 10.1128/JVI.02011-09. PubMed PMID: 19955314; PMCID: PMC2812394.

Thorne LG, Bouhaddou M, Reuschl AK, Zuliani-Alvarez L, Polacco B, Pelin A, Batra J, Whelan MVX, Ummadi M, Rojc A, Turner J, Obernier K, Braberg H, Soucheray M, Richards A, Chen KH, Harjai B, Memon D, Hosmillo M, Hiatt J, Jahun A, Goodfellow IG, Fabius JM, Shokat K, Jura N, Verba K, Noursadeghi M, Beltrao P, Swaney DL, Garcia-Sastre A, Jolly C, Towers GJ, Krogan NJ. Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant. bioRxiv. 2021. doi: 10.1101/2021.06.06.446826. PubMed PMID: 34127972; PMCID: PMC8202424.

Guo K, Barrett BS, Mickens KL, Hasenkrug KJ, Santiago ML. Interferon Resistance of Emerging SARS-CoV-2 Variants. bioRxiv. 2021. doi: 10.1101/2021.03.20.436257. PubMed PMID: 33758840; PMCID: PMC7986999.

Jiang HW, Zhang HN, Meng QF, Xie J, Li Y, Chen H, Zheng YX, Wang XN, Qi H, Zhang J, Wang PH, Han ZG, Tao SC. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cell Mol Immunol. 2020;17(9):998-1000. doi: 10.1038/s41423-020-0514-8. PubMed PMID: 32728199; PMCID: PMC7387808.

Miorin L, Kehrer T, Sanchez-Aparicio MT, Zhang K, Cohen P, Patel RS, Cupic A, Makio T, Mei M, Moreno E, Danziger O, White KM, Rathnasinghe R, Uccellini M, Gao S, Aydillo T, Mena I, Yin X, Martin-Sancho L, Krogan NJ, Chanda SK, Schotsaert M, Wozniak RW, Ren Y, Rosenberg BR, Fontoura BMA, Garcia-Sastre A. SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling. Proc Natl Acad Sci USA. 2020;117(45):28344-54. doi: 10.1073/pnas.2016650117. PubMed PMID: 33097660; PMCID: PMC7668094.

Oh SJ, Shin OS. SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response. Cells. 2021;10(3). doi: 10.3390/cells10030530. PubMed PMID: 33801464; PMCID: PMC7999926.

Parker MD, Lindsey BB, Shah DR, Hsu S, Keeley AJ, Partridge DG, Leary S, Cope A, State A, Johnson K, Ali N, Raghei R, Heffer J, Smith N, Zhang P, Gallis M, Louka SF, Whiteley M, Foulkes BH, Christou S, Wolverson P, Pohare M, Hansford SE, Green LR, Evans C, Raza M, Wang D, Gaudieri S, Mallal S, , de Silva TI. Altered Subgenomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections. bioRxiv. 2021.

Earle KA, Ambrosino DM, Fiore-Gartland A, Goldblatt D, Gilbert PB, Siber GR, Dull P, Plotkin SA. Evidence for antibody as a protective correlate for COVID-19 vaccines. Vaccine. 2021;39(32):4423-8. doi: 10.1016/j.vaccine.2021.05.063. PubMed PMID: 34210573; PMCID: PMC8142841.

Khoury DS, Cromer D, Reynaldi A, Schlub TE, Wheatley AK, Juno JA, Subbarao K, Kent SJ, Triccas JA, Davenport MP. Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection. Nat Med. 2021;27(7):1205-11. doi: 10.1038/s41591-021-01377-8. PubMed PMID: 34002089.

Bergwerk M, Gonen T, Lustig Y, Amit S, Lipsitch M, Cohen C, Mandelboim M, Gal Levin E, Rubin C, Indenbaum V, Tal I, Zavitan M, Zuckerman N, Bar-Chaim A, Kreiss Y, Regev-Yochay G. Covid-19 Breakthrough Infections in Vaccinated Health Care Workers. N Engl J Med. 2021. doi: 10.1056/NEJMoa2109072. PubMed PMID: 34320281.