Like cellular DNA, cellular and non-cellular agents constantly damage RNA. In DNA, many lesions can block replication. The 3′ → 5′ exonuclease proofreading activity of replicative DNA polymerases reduces the misincorporation of incorrect nucleotides. Proofreading activities of replicative DNA polymerases participate in three distinct accuracy mechanisms: proofreading, mismatch repair, and Okazaki fragment maturation.
RNA virus replication has a high error rate resulting in a diverse population of genome mutants, also known as “quasispecies.” For the adaption and to thrive in host cells, RNA viruses need to maintain an intact and replication-competent genome. Coronaviruses need to achieve this with a more extensive and complex genome. A typical coronavirus genome can be as large as 32 kilobases of positive-sense RNA.
Coronavirus Replication Mechanism for SARS-CoV and SARS-CoV-2
To enter the host cell,
(1) the coronavirus binds to the ACE2 receptor to initiate the viral entry,
(2) internalizes the vacuole containing the virus, and
(3) the membrane fuses with the virus to
(4) release it into the cytoplasm of the host cell.
(5) The genome is then translated to produce the polyproteins pp1a and pp1ab, which are cleaved by proteases to yield the 16 non-structural proteins (NSPs) that form the RNA replicase-transcriptase complex.
(6) The Viral genome is duplicated, and mRNA encoding structural proteins are transcribed.
(7) The subgenomic mRNAs are translated into structural proteins.
(8) Formation of the new virion occurs on modified intracellular membranes derived from the rough endoplasmic reticulum (ER) in the perinuclear region.
(9) Finally, the new virion is released.
Coronaviruses are known to encode a proofreading function in the non-coding protein nsp14 to minimize transcription errors and mutation rates.
Genetic inactivation of the 3’-to-5’- exonuclease (ExoN) proofreading activity in engineered coronavirus genomes resulted in increased mutation rates. The increase in mutation rates was approximately 15- to 20-fold. These findings indicated that ExoN activity is essential for the replication fidelity of the virus. Coronaviruses encode a 3’-to-5’-exoribonuclease activity (ExoN) in the nonstructural protein 14 (nsp14).
Nsp14 is a 60 kDa enzyme with an N-terminal exonuclease (ExoN) domain and a C-terminal N7-methyltransferase (N7-MTase) domain. The ExoN domain appears to be responsible for replication fidelity, and the N7-MTase domain is involved in mRNA capping. Nsp14 is also involved in several other virus life cycle processes and pathogenicity, including innate immune responses and viral genome recombination. The ExoN domain seems to correct errors made by the RNA-dependent RNA polymerase (RdRp) by removing mismatched nucleotides from the 3′ end of the growing RNA strand. In 2015, Ma et al. solved the structure of SARS-CoV nsp14–nsp10 heterodimer complexes to reveal the methyl transfer mechanism of the nsp14-mTase domain. The structure showed that methyl receptor guanosine-P3-adenosine-5’,5’-triphosphate (GpppA) binds near S-adenosyl methionine (SAM) in the complex.
Figure 1: Two views of the nsp14–nsp10-SAH-GpppA heterodimer structure. The ligands are shown as spheres and are highlighted in color.
Ma et al. solved the structure of the nsp14–nsp10 complex by coexpressing the full-length nsp14 protein with the nsp10 protein in Escherichia coli and purifying the preformed complex. The research group refined the unliganded, SAM-bound, and S-adenosyl homocysteine (SAH)–guanosine-P3-adenosine-5′,5′-triphosphate (GpppA)–bound nsp14–nsp10 complex structures to 3.4 Å, 3.2 Å, and 3.3 Å resolutions, respectively.
Nsp14 is highly conserved within the Coronaviridae family. The nsp14-nsp10 complex excises 3’-mismatched nucleotides from double-stranded RNA. This ExoN activity of the protein complex results in a low mutation rate for the coronavirus. A DEDDh (a five catalytic amino acid) motif drives catalysis by nsp14, necessary for viral replication and transcription.
Nsp14 functions as S-adenosyl methionine (SAM)-dependent (guanine-N7) methyltransferase (N7-MTase), and the assembly of a cap1 structure at the 5′ end of viral mRNA assists in translation and evading host defense.
The formation of the cap in SARS-CoV requires four sequential reactions.
(1) First, nsp13 RNA triphosphatase (RTPase) hydrolyzes nascent RNA to yield pp-RNA.
(2) An unknown guanylyltransferase (GTase) hydrolyzes GTP, transfers the product GMP to pp-RNA, and creates Gppp-RNA.
(3) The nsp14 methylates the 5′ guanine of the Gppp-RNA at the N7 position,
(4) followed by methylation of the ribose of the first nucleotide at the 2′-O-position by nsp16.
Nsp10 is known to activate the 2′-O–MTase activity of nsp16 by stabilizing the SAM-binding pocket and extending the substrate RNA-binding groove of nsp16. Similarly, the ExoN activity of nsp14 is fully unleashed only in the presence of nsp10.
However, the exact molecular mechanism of this activation is poorly understood.
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Fazlieva R, Spittle CS, Morrissey D, Hayashi H, Yan H, Matsumoto Y. Proofreading exonuclease activity of human DNA polymerase delta and its effects on lesion-bypass DNA synthesis. Nucleic Acids Res. 2009 May;37(9):2854-66. doi: 10.1093/nar/gkp155. Epub 2009 Mar 12. PMID: 19282447; PMCID: PMC2685094. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685094/
Ma Y, Wu L, Shaw N, Gao Y, Wang J, Sun Y, Lou Z, Yan L, Zhang R, Rao Z. Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex. Proc Natl Acad Sci U S A. 2015 Jul 28;112(30):9436-41. doi: 10.1073/pnas.1508686112. Epub 2015 Jul 9. PMID: 26159422; PMCID: PMC4522806. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522806/
Robson F, Khan KS, Le TK, Paris C, Demirbag S, Barfuss P, Rocchi P, Ng WL. Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting. Mol Cell. 2020 Sep 3;79(5):710-727. doi: 10.1016/j.molcel.2020.07.027. Epub 2020 Aug 4. Erratum in: Mol Cell. 2020 Dec 17;80(6):1136-1138. PMID: 32853546; PMCID: PMC7402271.
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