Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Czech Republic

Department of Biotechnology University of Chemistry and Technology Prague Czech Republic

See more in PubMed

Bakhshandeh B, Jahanafrooz Z, Abbasi A, Goli MB, Sadeghi M, Mottaqi MS, et al. Mutations in SARS‐CoV‐2; consequences in structure, function, and pathogenicity of the virus. Microb Pathog. 2021;154:104831. 10.1016/j.micpath.2021.104831 PubMed DOI PMC

Barakat K, Ahmed M, Tabana Y, Ha M. A ‘deep dive’ into the SARS‐Cov‐2 polymerase assembly: identifying novel allosteric sites and analyzing the hydrogen bond networks and correlated dynamics. J Biomol Struct Dyn. 2022;40(19):9443–9463. 10.1080/07391102.2021.1930162 PubMed DOI

Biswal M, Diggs S, Xu D, Khudaverdyan N, Lu J, Fang J, et al. Two conserved oligomer interfaces of NSP7 and NSP8 underpin the dynamic assembly of SARS‐CoV‐2 RdRP. Nucleic Acids Res. 2021;49(10):5956–5966. 10.1093/nar/gkab370 PubMed DOI PMC

Carabelli AM, Peacock TP, Thorne LG, Harvey WT, Hughes J, Peacock SJ, et al. SARS‐CoV‐2 variant biology: immune escape, transmission and fitness. Nat Rev Microbiol. 2023;21(3):162–177. 10.1038/s41579-022-00841-7 PubMed DOI PMC

Courouble VV, Dey SK, Yadav R, Timm J, Harrison JJEK, Ruiz FX, et al. Revealing the structural plasticity of SARS‐CoV‐2 nsp7 and nsp8 using structural proteomics. J Am Soc Mass Spectrom. 2021;32(7):1618–1630. 10.1021/jasms.1c00086 PubMed DOI PMC

Füzik T, Ulbrich P, Ruml T. Efficient mutagenesis independent of ligation (EMILI). J Microbiol Methods. 2014;106:67–71. 10.1016/j.mimet.2014.08.003 PubMed DOI

Gao Y, Yan LM, Huang YC, Liu FJ, Zhao Y, Cao L, et al. Structure of the RNA‐dependent RNA polymerase from COVID‐19 virus. Science. 2020;368(6492):779–782. 10.1126/science.abb7498 PubMed DOI PMC

Goldswain H, Dong X, Penrice‐Randal R, Alruwaili M, Shawli GT, Prince T, et al. The P323L substitution in the SARS‐CoV‐2 polymerase (NSP12) confers a selective advantage during infection. Genome Biol. 2023;24(1):47. 10.1186/s13059-023-02881-5 PubMed DOI PMC

Gorbalenya AE, Enjuanes L, Ziebuhr J, Snijder EJ. Evolving the largest RNA virus genome. Virus Res. 2006;117(1):17–37. 10.1016/j.virusres.2006.01.017 PubMed DOI PMC

Hillen HS, Kokic G, Farnung L, Dienemann C, Tegunov D, Cramer P. Structure of replicating SARS‐CoV‐2 polymerase. Nature. 2020;584(7819):154–156. 10.1038/s41586-020-2368-8 PubMed DOI

Jena NR, Pant S. Peptide inhibitors derived from the nsp7 and nsp8 cofactors of nsp12 targeting different substrate binding sites of nsp12 of the SARS‐CoV‐2. J Biomol Struct Dyn. 2023;11:1–13. 10.1080/07391102.2023.2235012 PubMed DOI

Jones AN, Mourão A, Czarna A, Matsuda A, Fino R, Pyrc K, et al. Characterization of SARS‐CoV‐2 replication complex elongation and proofreading activity. Sci Rep. 2022;12(1):9593. 10.1038/s41598-022-13380-1 PubMed DOI PMC

Kannan SR, Spratt AN, Quinn TP, Heng X, Lorson CL, Sönnerborg A, et al. Infectivity of SARS‐CoV‐2: there is something more than D614G? J Neuroimmune Pharmacol. 2020;15(4):574–577. 10.1007/s11481-020-09954-3 PubMed DOI PMC

Kim SM, Kim EH, Casel MAB, Kim YI, Sun R, Kwak MJ, et al. SARS‐CoV‐2 variants with NSP12 P323L/G671S mutations display enhanced virus replication in ferret upper airways and higher transmissibility. Cell Rep. 2023;42(9):113077. 10.1016/j.celrep.2023.113077 PubMed DOI PMC

Kirchdoerfer RN, Ward AB. Structure of the SARS‐CoV nsp12 polymerase bound to nsp7 and nsp8 co‐factors. Nat Commun. 2019;10:2342. 10.1038/s41467-019-10280-3 PubMed DOI PMC

Kwon JH, Kim JM, Lee DH, Park AK, Kim IH, Kim DW, et al. Genomic epidemiology reveals the reduction of the introduction and spread of SARS‐CoV‐2 after implementing control strategies in Republic of Korea, 2020. Virus Evol. 2021;7(2):veab077. 10.1093/ve/veab077 PubMed DOI PMC

Madru C, Tekpinar AD, Rosario S, Czernecki D, Brûlé S, Sauguet L, et al. Fast and efficient purification of SARS‐CoV‐2 RNA dependent RNA polymerase complex expressed in Escherichia coli. PLoS One. 2021;16(4):e0250610. 10.1371/journal.pone.0250610 PubMed DOI PMC

Magazine N, Zhang TY, Wu YY, McGee MC, Veggiani G, Huang WS. Mutations and evolution of the SARS‐CoV‐2 spike protein. Viruses‐Basel. 2022;14(3):640. 10.3390/v14030640 PubMed DOI PMC

Malone BF, Perry JK, Olinares PDB, Lee HW, Chen JM, Appleby TC, et al. Structural basis for substrate selection by the SARS‐CoV‐2 replicase. Nature. 2023;614(7949):781–787. 10.1038/s41586-022-05664-3 PubMed DOI PMC

McDonald SM. RNA synthetic mechanisms employed by diverse families of RNA viruses. WIREs RNA. 2013;4(4):351–367. 10.1002/wrna.1164 PubMed DOI PMC

Miropolskaya N, Kozlov M, Petushkov I, Prostova M, Pupov D, Esyunina D, et al. Effects of natural polymorphisms in SARS‐CoV‐2 RNA‐dependent RNA polymerase on its activity and sensitivity to inhibitors. Biochimie. 2023;206:81–88. 10.1016/j.biochi.2022.10.007 PubMed DOI PMC

Mutlu O, Ugurel OM, Sariyer E, Ata O, Inci TG, Ugurel E, et al. Targeting SARS‐CoV‐2 Nsp12/Nsp8 interaction interface with approved and investigational drugs: an in silico structure‐based approach. J Biomol Struct Dyn. 2022;40(2):918–930. 10.1080/07391102.2020.1819882 PubMed DOI PMC

Nakamoto S, Kanda T, Wu S, Shirasawa H, Yokosuka O. Hepatitis C virus NS5A inhibitors and drug resistance mutations. World J Gastroenterol. 2014;20(11):2902–2912. 10.3748/wjg.v20.i11.2902 PubMed DOI PMC

Ng TI, Correia I, Seagal J, DeGoey DA, Schrimpf MR, Hardee DJ, et al. Antiviral drug discovery for the treatment of COVID‐19 infections. Viruses‐Basel. 2022;14(5):961. 10.3390/v14050961 PubMed DOI PMC

Ogando NS, Zevenhoven‐Dobbe JC, van der Meer Y, Bredenbeek PJ, Posthuma CC, Snijder EJ. The enzymatic activity of the nsp14 exoribonuclease is critical for replication of MERS‐CoV and SARS‐CoV‐2. J Virol. 2020;94(23):e01246‐20. 10.1128/JVI.01246-20 PubMed DOI PMC

Pachetti M, Marini B, Benedetti F, Giudici F, Mauro E, Storici P, et al. Emerging SARS‐CoV‐2 mutation hot spots include a novel RNA‐dependent‐RNA polymerase variant. J Transl Med. 2020;18(1):179. 10.1186/s12967-020-02344-6 PubMed DOI PMC

Peng Q, Peng RC, Yuan B, Zhao JR, Wang M, Wang XX, et al. Structural and biochemical characterization of the nsp12‐nsp7‐nsp8 Core polymerase complex from SARS‐CoV‐2. Cell Rep. 2020;31(11):107774. 10.1016/j.celrep.2020.107774 PubMed DOI PMC

Piret J, Boivin G. Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management. Antimicrob Agents Chemother. 2011;55(2):459–472. 10.1128/Aac.00615-10 PubMed DOI PMC

Reshamwala SMS, Likhite V, Degani MS, Deb SS, Noronha SB. Mutations in SARS‐CoV‐2 nsp7 and nsp8 proteins and their predicted impact on replication/transcription complex structure. J Med Virol. 2021;93(7):4616–4619. 10.1002/jmv.26791 PubMed DOI PMC

Ruan Z, Liu C, Guo Y, He Z, Huang X, Jia X, et al. SARS‐CoV‐2 and SARS‐CoV: virtual screening of potential inhibitors targeting RNA‐dependent RNA polymerase activity (NSP12). J Med Virol. 2021;93(1):389–400. 10.1002/jmv.26222 PubMed DOI PMC

Sáez‐Alvarez Y, Arias A, del Aguila C, Agudo R. Development of a fluorescence‐based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs. Sci Rep. 2019;9:5397. 10.1038/s41598-019-41998-1 PubMed DOI PMC

Seidel SAI, Dijkman PM, Lea WA, van den Bogaart G, Jerabek‐Willemsen M, Lazic A, et al. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods. 2013;59(3):301–315. 10.1016/j.ymeth.2012.12.005 PubMed DOI PMC

Senthilazhagan K, Sakthimani S, Kallanja D, Venkataraman S. SARS‐CoV‐2: analysis of the effects of mutations in non‐structural proteins. Arch Virol. 2023;168(7):186. 10.1007/s00705-023-05818-2 PubMed DOI

Singer J, Gifford R, Cotten M, Robertson D. CoV‐GLUE: a web application for tracking SARS‐CoV‐2 genomic variation. Preprints; 2020. 10.20944/preprints202006.0225.v1 DOI

Snijder EJ, Decroly E, Ziebuhr J. The nonstructural proteins directing coronavirus RNA synthesis and processing. Adv Virus Res. 2016;96:59–126. 10.1016/bs.aivir.2016.08.008 PubMed DOI PMC

Song ZQ, Xu YF, Bao LL, Zhang L, Yu P, Qu YJ, et al. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses‐Basel. 2019;11(1):59. 10.3390/v11010059 PubMed DOI PMC

Stern A, Andino R. Chapter 17 ‐ viral evolution: it is all about mutations. In: Katze MG, Korth MJ, Law GL, Nathanson N, editors. Viral pathogenesis. Third ed. Academic Press; 2016. p. 233–240. 10.1016/B978-0-12-800964-2.00017-3 DOI

Subissi L, Posthuma CC, Collet A, Zevenhoven‐Dobbe JC, Gorbalenya AE, Decroly E, et al. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci U S A. 2014;111(37):E3900–E3909. 10.1073/pnas.1323705111 PubMed DOI PMC

Treviño MA, Pantoja‐Uceda D, Laurents D, Mompeán M. SARS‐CoV‐2 Nsp8 N‐terminal domain folds autonomously and binds dsRNA. Nucleic Acids Res. 2023;51:10041–10048. 10.1093/nar/gkad714 PubMed DOI PMC

Walker AP, Fan HT, Keown JR, Knight ML, Grimes JM, Fodor E. The SARS‐CoV‐2 RNA polymerase is a viral RNA capping enzyme. Nucleic Acids Res. 2021;49(22):13019–13030. 10.1093/nar/gkab1160 PubMed DOI PMC

Wang B, Svetlov V, Wolf YI, Koonin EV, Nudler E, Artsimovitch I. Allosteric activation of SARS‐CoV‐2 RNA‐dependent RNA polymerase by Remdesivir triphosphate and other phosphorylated nucleotides. MBio. 2021;12(3):e01423‐21. 10.1128/mBio.01423-21 PubMed DOI PMC

Weiss SR, Leibowitz JL. Coronavirus pathogenesis. Adv Virus Res. 2011;81(81):85–164. 10.1016/B978-0-12-385885-6.00009-2 PubMed DOI PMC

White KL, Margot NA, Wrin T, Petropoulos CJ, Miller MD, Naeger LK. Molecular mechanisms of resistance to human immunodeficiency virus type 1 with reverse transcriptase mutations K65R and K65R+M184V and their effects on enzyme function and viral replication capacity. Antimicrob Agents Chemother. 2002;46(11):3437–3446. 10.1128/Aac.46.11.3437-3446.2002 PubMed DOI PMC

WHO . WHO Coronavirus (COVID‐19) dashboard [Dashboard] . 2023a. [cited 2024 Jan 2]. Available from https://data.who.int/

WHO . Statement on the Fifteenth Meeting of the International Health Regulations (2005) Emergency Committee Regarding the Coronavirus Disease (COVID‐19) Pandemic . 2023b. [cited 2023 Dec 18]. Available from https://www.who.int/news/item/05‐05‐2023‐statement‐on‐the‐fifteenth‐meeting‐of‐the‐international‐health‐regulations‐(2005)‐emergency‐committee‐regarding‐the‐coronavirus‐disease‐(covid‐19)‐pandemic?adgroupsurvey=%7Badgroupsurvey%7D&gclid=EAIaIQobChMI4Ojtsdbe_gIVjQRyCh07igt4EAAYASACEgJ9pfD_BwE&fbclid=IwAR2M8EAyiSrAodhK9p‐X582nHkP2AigpSX8pYIsLsPwqYh4SG26RGokGe7E

Wilamowski M, Hammel M, Leite W, Zhang Q, Kim Y, Weiss KL, et al. Transient and stabilized complexes of Nsp7, Nsp8, and Nsp12 in SARS‐CoV‐2 replication. Biophys J. 2021;120(15):3152–3165. 10.1016/j.bpj.2021.06.006 PubMed DOI PMC

Wu AP, Peng YS, Huang BY, Ding X, Wang XY, Niu PH, et al. Genome composition and divergence of the novel coronavirus (2019‐nCoV) originating in China. Cell Host Microbe. 2020;27(3):325–328. 10.1016/j.chom.2020.02.001 PubMed DOI PMC

Xiao YB, Ma QJ, Restle T, Shang WF, Svergun DI, Ponnusamy R, et al. Nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 Heterotrimer that exhibits primer‐independent RNA polymerase activity. J Virol. 2012;86(8):4444–4454. 10.1128/Jvi.06635-11 PubMed DOI PMC

Xu JX, Jiang X, Zhang YL, Dong Y, Ma CL, Jiang HQ, et al. Multiscale characterization reveals oligomerization dependent phase separation of primer‐independent RNA polymerase nsp8 from SARS‐CoV‐2. Commun Biol. 2022;5(1):925. 10.1038/s42003-022-03892-x PubMed DOI PMC

Xu XY, Chen YH, Lu XY, Zhang WL, Fang WX, Yuan LP, et al. An update on inhibitors targeting RNA‐dependent RNA polymerase for COVID‐19 treatment: promises and challenges. Biochem Pharmacol. 2022;205:115279. 10.1016/j.bcp.2022.115279 PubMed DOI PMC

Yin XY, Popa H, Stapon A, Bouda E, Garcia‐Diaz M. Fidelity of ribonucleotide incorporation by the SARS‐CoV‐2 replication complex. J Mol Biol. 2023;435(5):167973. 10.1016/j.jmb.2023.167973 PubMed DOI PMC

Zhai YJ, Sun F, Li XM, Pang H, Xu XL, Bartlam M, et al. Insights into SARS‐CoV transcription and replication from the structure of the nsp7‐nsp8 hexadecamer. Nat Struct Mol Biol. 2005;12(11):980–986. 10.1038/nsmb999 PubMed DOI PMC

Zhang C, Li L, He J, Chen C, Su D. Nonstructural protein 7 and 8 complexes of SARS‐CoV‐2. Protein Sci. 2021;30(4):873–881. 10.1002/pro.4046 PubMed DOI PMC