RNA Dependent RNA Polymerases of Severe Acute Respiratory Syndrome-Related Coronaviruses- An Insight into their Active Sites and Mechanism of Action

Main Article Content

Peramachi Palanivelu

Abstract

Aim: To analyze the RNA-dependent RNA polymerases (RdRps) of Severe Acute Respiratory Syndrome (SARS)-related coronaviruses (CoVs) to find out the conserved motifs, metal binding sites and catalytic amino acids and propose a plausible mechanism of action for these enzymes, using SARS-CoV-2 RdRp as a model enzyme.

Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM), X-ray crystallographic and cryo-Electron microscopic (cryo-EM) data were analyzed.

Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis, X-ray crystallographic and cryo-EM data of these enzymes from RNA viral pathogens were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the RdRps.

Results: Multiple sequence alignment (MSA) of RdRps from different SARS-related CoVs show a large number of highly conserved motifs among them. Though the RdRp from the Middle Eastern Respiratory syndrome (MERS)-CoV differed in many conserved regions yet the active site regions are completely conserved. Possible catalytic regions consist of an absolutely conserved amino acid K, as in single subunit (SSU) RNA polymerases and most of the DNA dependent DNA polymerases (DdDps). The invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU DNA dependent RNA polymerases (DdRps), prokaryotic multi-subunit (MSU) DdRps and DdDps is also highly conserved in the RdRps of SARS-CoVs. The universal metal binding motif –GDD- and an additional motif–SDD- are also found in all SARS-CoV RdRps. In stark contrast, the (–) strand RNA viral pathogens like Ebola, rabies, etc. use –GDN- rather than –GDD- for catalytic metal binding. An invariant YA pair (instead of an YG pair) is found in the primases of the SARS-CoVs. The SARS-CoVs RdRps and primases exhibit very similar active site and catalytic regions with almost same distance conservations between the template binding YG/YA pair and the catalytic K. In SARS-CoV RdRps an invariant R is placed at -5 which is shown to play a role in nucleoside triphosphate (NTP) selection and is in close agreement with SSU DdRps (viral family) and DdDps. In primases no such invariant R/K/H is found very close to the catalytic K in the downstream region, as found in RdRp and Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domains. An invariant YA pair is placed in the NiRAN domain instead of an YG pair, and an invariant H is placed at -5 position. Moreover, the Zn binding motif with the completely conserved Cs and a few DxD/DxxD type metal binding motifs are found in the RdRps and NiRAN domain. However, the primases contained only the DXD type metal binding motifs.

Conclusions: The SARS and SARS-related CoV RdRps are very similar as large peptide regions are highly conserved among them. The closer identity between the RdRps of palm civet-CoV and SARS-CoV (CoV-1) suggest their possible link as found between their spike proteins also. The invariant YG and KL pairs may play a role in template binding and catalysis in SARS-CoV RdRps as reported in DdDps. An additional invariant –YAN- motif found in SARS-CoV RdRps may play a crucial role in nucleotide discrimination.

Keywords:
Coronaviruses, RNA-dependent RNA polymerases, primases, NiRAN domain, polymerase conserved motifs, active sites, catalytic mechanism.

Article Details

How to Cite
Palanivelu, P. (2020). RNA Dependent RNA Polymerases of Severe Acute Respiratory Syndrome-Related Coronaviruses- An Insight into their Active Sites and Mechanism of Action. International Journal of Biochemistry Research & Review, 29(10), 29-52. https://doi.org/10.9734/ijbcrr/2020/v29i1030236
Section
Original Research Article

References

Kenneth KS Ng, Jamie J. Arnold, and Craig E. Cameron. Structure-function relationships among RNA-Dependent RNA polymerases. Curr Top Microbiol Immunol. 2008;320:137–156.

Wang Q, Wu J, Wang H, Gao Y, Liu Q, Mu A, Ji W, et al. Structural basis for RNA replication by the SARS-CoV-2 polymerase. Cell. 2020;182:417–428.

Alberts B, Johnson J, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell. New York: Garland Science; 2003.

Flint SJ, Enquist LW, Racaniello VR, Skalka AM. Principles of virology: molecular biology, pathogenesis, and control of animal viruses. Molecular biology. Washington, DC: ASM Press; 2004.

Elena SF, Sanjuan R. Adaptive value of high mutation rates of RNA viruses: Separating causes from consequences. J. Virol. 2005;79:11555–11558.

Castro C, Arnold JJ, Cameron CE. Incorporation fidelity of the viral RNA-dependent RNA polymerase: a kinetic, thermodynamic and structural perspective. Virus Res. 2005;107:141–149.

Palanivelu P. Analyses of the Spike Proteins of Severe Acute Respiratory Syndrome-Related Coronaviruses. Microbiol Res J Int. 2020; 30:32-50.

Chan JFW, Kok KH, Zhu Z, Chu H , To KKW , Yuan S , Yuen KY.. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect. 2020;9:221-236.

McBride R, Fielding BC. The role of severe acute respiratory syndrome (SARS)-coronavirus accessory proteins in virus pathogenesis. Viruses. 2012;4:2902-2923.

Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. In Maier HJ, Bickerton E, Britton P, editors. Coronaviruses. Met Mol Biol. 1282. Springer. 2015;1–23.
DOI: 10.1007/978-1-4939-2438-7_1.

Thoms M, Buschauer R, Ameismeier M, Koepke L, Denk T, Hirschenberger M. Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science. 2020;369:1249–1255.

Wolff G, Limpens RWAL, Zevenhoven-Dobbe JC, Laugks U, Zheng S, de Jong AWM, Koning RI, et al. A molecular pore spans the double membrane of the coronavirus replication organelle. Science. 2020;369:1395-1398.

Lei J, Kusov Y, Hilgenfeld R. Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein. Antiviral Res. 2018;149:58–74.

Sparrer KM, Gack MU. Intracellular detection of viral nucleic acids. Curr. Opin. Microbiol. 2015;26:1-9.

Bianchi M, Benvenuto D, Giovanetti M, Angeletti S, Ciccozzi M, Pascarella S.. Sars-CoV-2 Envelope and Membrane Proteins: Structural differences Linked to Virus Characteristics? BioMed Res. Int. 2020;2020:1-6.

Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579:265-269.

Hillen HS, Kokic G, Farnung L, Dienemann C, Tegunov D, Cramer P. Structure of replicating SARS-CoV-2 polymerase. bioRxiv preprint; 2020. Available:doi.org/10.1101/2020.04.27.063180.

Subissi L, et al. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets. Antiviral Res. 2014;101:122-130.
DOI: 10.1016/j.antiviral.2013.11.006.

Gao Y, Yan L, Huang Y, Liu F, Zhao Y, Cao L, Wang T, et al. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. 2020;368:779-782.

Zhang WF, Stephen P, Thériault JF, Wang R, Lin SX. Novel coronavirus polymerase and nucleotidyl-transferase structures: potential to target new outbreaks. J Phys Chem Lett. 2020;11:4430-4435.

Frediansyah A, Tiwari R Sharun K, Dhama K, Harapan H. Antivirals for COVID-19: A critical review. Clinical Epidemiology and Global Health; 2021.
doi.org/10.1016/j.cegh.2020.07.006

Palanivelu P. DNA polymerases – An insight into their active sites and mechanism of action, In: Recent Advances in Biological Research. Sciencedomain International Book Publishers, UK. 2019;1: 1-39,
ISBN: 978-81-934224-4-1,
DOI:10.9734/bpi/rabr/v1,

Palanivelu P. Single Subunit RNA Polymerases: An Insight into their Active Sites and Catalytic Mechanism, In: Advances and Trends in Biotechnology and Genetics. 2019;1:1-38.

Sciencedomain International Book Publishers, UK.
ISBN: 978-93-89246-59-9,
DOI:10.9734/bpi/atbg/v1,

Palanivelu P. Multi-subunit RNA Polymerases of Bacteria - An insight into their active sites and catalytic mechanism. Indian J Sci Technol. 2018;11:1-37.

Palanivelu, P. Eukaryotic multi-subunit DNA dependent RNA Polymerases: An insight into their active sites and catalytic mechanism. In: Emerging Trends and Research in Biological Science. Sciencedomain International Book Publishers, UK. 2020;1:1-66.
ISBN: 978-93-89562-56-9,
DOI: 10.9734/bpi/etrbs/v1,

Palanivelu, P. Active sites of the multi-subunit RNA polymerases of eubacteria and chloroplasts are similar in structure and function: Recent perspectives. In: Current Research Trends in Biological Science. Sciencedomain International Book Publishers, UK. 2020;2:26-61.
ISBN: 978-93-90149-66-7,
DOI: 10.9734/bpi/crtbs/v2,

Hansen JL, Long AM, Schultz SC. Structure of the RNA-dependent RNA polymerase of poliovirus. Structure. 1997; 5:1109-1127.

Sangita V, Prasad BVLS, Selvarajan R. RNA dependent RNA polymerases: Insights from structure, function and evolution. Viruses. 2018;10:76-99.

Sankar S, Porter AG. Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli. DNA polymerase I. J Biol Chem. 1992; 267:10168–10176.

Jablonski SA, Luo M, Morrow CD. Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. J Virol. 1991;6:4565-4572.

Inokuchi Y, Hirashima A. Interference with viral infection by RNA replicase deleted at the carboxy-terminal region. J. Virol. 1987; 61:3946-3949.

Yap TL, Xu T, Chen YL, Malet H, Egloff MP, Canard B, Vasudevan SG, Lescar J. Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-Angstrom resolution. J Virol. 2007;81: 4753–4765.

Lehmann KC, Gulyaeva A, Zevenhoven-Dobbe JC, Janssen GMC, Ruben M, Overkleeft HS, van Veelen PA, Samborskiy DV, Kravchenko AA, Leontovich AM, et al.. Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses. Nucleic Acids Res. 2015;43:8416–8434.

Kostyuk SM, Dragan DL, Lyakhov VO, Rechinsky VL, Tunitskaya BK, Chernov SN, Kochetkov E. Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA. FEBS Letters. 1995; 369:165-168.

Imbert I, Guillemot JC, Bourhis JM, Bussetta C, Coutard B, Egloff MP, Ferron F. et al. A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. EMBO J. 2006;25:4933-4942.

te Velthuis AJW, van den Worm SH, Snijder EJ. The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic Acids Res. 2012;40:1737-17.

Subissia L, Posthumab CC, Colleta A, Dobbeb JCZ, Gorbalenyab AE, Decrolya E, Snijderb EJ, Canarda B, Imberta I, One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proc Natl Acad Sci (USA). 2014;E3900–E3909.

Schmidt ML, Hoenen T. Characterization of the catalytic center of the Ebola virus L polymerase. PLoS Negl Trop Dis. 2017;11:1-14.

Tchesnokov EP, Raeisimakiani P, Ngure M, Marchant D, Götte M. Recombinant RNA-dependent RNA polymerase complex of ebola virus. Sci Rep. 2018;8:3970-3979.

Zhang G, Campbell EA, Minakhin L, Richter C, Severinov K, Darst SA. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell. 1999; 98:811-824.

Woody AY, Eaton SS, Osumi-Davis PA, Woody RW, Asp537 and Asp812 in bacteriophage T7 RNA polymerase as metal ion-binding sites studied by EPR, flow-dialysis, and transcription. Biochemistry. 1996;35:144-152.