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RNA-dependent RNA polymerase/ RNA-directed RNA polymerase (RdRp)/ nsp12
Function
Responsible for replication and transcription of the viral RNA genome.[1][2]
Together with the proteins nsp7 and a dimer of nsp8, nsp12 forms the . Without its co-factors, nsp12 has a low efficiency in polymerase activity. This complex mediates the RNA synthesis of the virus and is therefore an important part in the replication procedure[3].
Disease
The global COVID-19 pandemic, which started in 2019, is caused by the SARS-CoV-2.
Structure
Nsp12 consists of three domains, the nidoviruses specific N-terminal nidovirus RdRp-associated nucleotidyltransferase (D60-R249), the C-terminal (S367-F920) as well as an (A250-R365) connecting both[4]. At the N-terminus a beta-hairpin motif binds at the interface between the palm subdomain and the NiRAN domain[3].
The catalytic domain is a RdRp domain, and is described as a , consisting of three subdomains: the finger, palm and thumb. Together with the thumb, the long extended finger forms a closed-ring structure. It contains the motifs A-F that are highly conserved among viral RdRps and motif G [3]. Motifs A-E compose the active site at the palm subdomain, with motif C binding to the 3’ end of the RNA template. The motifs F and G are located in the fingers and are responsible for the positioning of the RNA template, while the first turn of the RNA is bound between the fingers and the thumb[5]. The fingertip consists of the motif F, interacting with the thumb subdomain and the finger extension loop. The finger extension loops, which themselves are supported by interactions with the nsp7-nsp8.1 heterodimer, stabilize the fingertip loop[3].
The main part of the interaction of the nsp7-nsp8 complex with nsp12 has the nsp7 above the thumb subdomain, while it stabilizes the conformation of the finger extension loops. The second nsp8 (nsp8.2) binds to the top of the finger subdomain and the interface domain and forms a significantly different conformation than nsp8.1[3].
For entering the catalytic chamber, the RNA gets guided through the template entrance, stabilized by the fingertip and finger extension loops. At the back side of the palm subdomain is a channel for the nucleotide triphosphate to enter the active site. The RNA duplex, made from template and product, can exit at the front of the polymerase, and has later to be separated in further steps by other nsps in order to function[3].
Studies suggest an interaction between the nsp12 and the helicase nsp13 (PDB: 6XEZ), as stable complexes can be formed of these two proteins. The helicase in complex with the replication-transcription complex might play a role in possible backtracking[6].
The to date published structures of complexes with nsp12 include 6M71, 6NUR, 6BTF, 7C2K, 7BZF, 7BV2, 6XEZ, 6XQB, 7CTT, 7BV1, 7BW4 and 6YYT.
Variations
The sequences of the nsp12 in SARS-CoV and SARS-CoV-2 have a high degree of identity (95.8%[7]) as well as a high similarity in their structure. The amino acid substitutions occur in all of the three subunits, but are not part of the active site or the interfaces between subunits of the polymerase.
A study on their enzymatic behavior showed a much lower efficiency of the SARS-CoV-2 nsp12-nsp7-nsp8 complex of around 35% compared to SARS-CoV. A cross-combination analysis of the different proteins of both viruses showed that the variations in nsp12 and nsp8 are the cause of the negative effect on the polymerase activity.
Further a comparison on the thermostability of the proteins showed a lower melting temperature for nsp12 and nsp8 compared to those of SARS-CoV[3].
Relevance
Through its essential role in the virus’s life cycle, the polymerase complex, especially nsp12, is the primary target for studies on nucleotide analog antiviral inhibitors. An example is remdesivir, an adenosine nucleoside triphosphate analog, acting with a delayed chain termination mechanism. It has been authorized as a drug for emergency use by the US Food and Drug Administration[6].
See also
Coronavirus_Disease 2019 (COVID-19)
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References
- ↑ Modeling of the SARS-COV-2 Genome
- ↑ Zhang C, Zheng W, Huang X, Bell EW, Zhou X, Zhang Y. Protein Structure and Sequence Reanalysis of 2019-nCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1. J Proteome Res. 2020 Apr 3;19(4):1351-1360. doi: 10.1021/acs.jproteome.0c00129., Epub 2020 Mar 24. PMID:32200634 doi:http://dx.doi.org/10.1021/acs.jproteome.0c00129
- ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 Peng Q, Peng R, Yuan B, Zhao J, Wang M, Wang X, Wang Q, Sun Y, Fan Z, Qi J, Gao GF, Shi Y. Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2. Cell Rep. 2020 Jun 16;31(11):107774. doi: 10.1016/j.celrep.2020.107774. Epub 2020, May 30. PMID:32531208 doi:http://dx.doi.org/10.1016/j.celrep.2020.107774
- ↑ Gao Y, Yan L, Huang Y, Liu F, Zhao Y, Cao L, Wang T, Sun Q, Ming Z, Zhang L, Ge J, Zheng L, Zhang Y, Wang H, Zhu Y, Zhu C, Hu T, Hua T, Zhang B, Yang X, Li J, Yang H, Liu Z, Xu W, Guddat LW, Wang Q, Lou Z, Rao Z. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. 2020 Apr 10. pii: science.abb7498. doi: 10.1126/science.abb7498. PMID:32277040 doi:http://dx.doi.org/10.1126/science.abb7498
- ↑ Hillen HS, Kokic G, Farnung L, Dienemann C, Tegunov D, Cramer P. Structure of replicating SARS-CoV-2 polymerase. Nature. 2020 May 21. pii: 10.1038/s41586-020-2368-8. doi:, 10.1038/s41586-020-2368-8. PMID:32438371 doi:http://dx.doi.org/10.1038/s41586-020-2368-8
- ↑ 6.0 6.1 Chen J, Malone B, Llewellyn E, Grasso M, Shelton PMM, Olinares PDB, Maruthi K, Eng ET, Vatandaslar H, Chait BT, Kapoor TM, Darst SA, Campbell EA. Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex. Cell. 2020 Jul 28. pii: S0092-8674(20)30941-7. doi: 10.1016/j.cell.2020.07.033. PMID:32783916 doi:http://dx.doi.org/10.1016/j.cell.2020.07.033
- ↑ Zhang L, Zhou R. Structural Basis of the Potential Binding Mechanism of Remdesivir to SARS-CoV-2 RNA-Dependent RNA Polymerase. J Phys Chem B. 2020 Aug 13;124(32):6955-6962. doi: 10.1021/acs.jpcb.0c04198. Epub, 2020 Jun 23. PMID:32521159 doi:http://dx.doi.org/10.1021/acs.jpcb.0c04198
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