Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

Abstract

The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3'-5' exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world.

Keywords: Capping; Proofreading; Replication; SARS coronavirus.

Fig. 1

Coronavirus genome replication and transcription. The two open reading frames (ORFs) of the genomic mRNA are translated by a cap-dependent mechanism, yielding two polyproteins, pp1a and pp1ab, the latter requiring −1 ribosomal frameshifting (FS) near the 3′ end of ORF1a. Subsequently, the two polyproteins are cleaved by two or three ORF1a-encoded proteases to release a total of 16 nonstructural proteins (nsp1–nsp16). Then, they assemble with modified intracellular membranes (Knoops et al., 2008) to form the replication/transcription complex (RTC). This huge RTC not only entails the synthesis of new genomes, but also produces a nested set of subgenomic (sg) mRNAs encoding structural and accessory proteins. The gray ball represents the cap structure. Nonstructural, structural and accessory genes are represented in blue, green and purple, respectively.

Fig. 2

Coronavirus nsp7/nsp8 structures. (A) Structure of the SARS-CoV nsp7/nsp8 hexadecamer supercomplex (from Zhai et al., 2005; PDB 2AHM). To the left of this panel, SARS-CoV nsp7 and the two conformations of nsp8 are colored in orange, cyan and dark blue, respectively. To the right of this panel, the surface is colored according to electrostatic potential (blue, positive charge; red, negative charge). (B) Structure of the FCoV nsp7/nsp8 heterotrimer (left panel): two nsp7 molecules (in green) are associated to one molecule of nsp8 (in pink) (from Xiao et al., 2012; PDB 3UBO). The right panel shows the surface colored according to electrostatic potential (blue, positive charge; red, negative charge). (C) C-terminal domain superimposition of the two SARS-CoV nsp8 forms (in cyan and dark blue) with FCoV nsp8 (in pink). The amino-terminal domain of nsp8 is very flexible, even in association with nsp7. Images were generated using PYMOL.

Fig. 3

Structure of 5′-end RNA cap. Cap structure is formed of a N7-methylated guanosine linked to the first RNA nucleotide via a 5′–5′ triphosphate bond. A second methyl group is added in 2′O position of the first nucleotide (for SARS-CoV, the first nucleotide is an adenosine).

Fig. 4

SARS-CoV RNA capping pathway. SARS-CoV uses a canonical capping pathway. The 5′-end γ-phosphate of the RNA is hydrolyzed by the nsp13 RTPase activity. Then, the still unknown RNA GTase transfers a GMP to the 5′-diphosphate RNA end, forming a 5′–5′ triphosphate bond. Next, the nsp14 N7-MTase transfers a methyl group from the SAM donor to the N7-position of the cap, forming the “cap-0” structure. Lastly, the nsp16/nsp10 2′O-MTase methylates the first RNA nucleotide in the 2′O position, to form the “cap-1” structure.

Fig. 5

Crystal structure of SARS-CoV nsp10/nsp16 complex. Ribbon representation of nsp10 (in gray) in complex with nsp16 (in yellow) (from Decroly et al., 2011; PDB 2XYQ). Mg²⁺ ion (in brown) found on nsp16 is localized at the opposite side of the 2′O-MTase active site which is formed by the catalytic tetrad K46, D130, K170 and E203 (in pink). The by-product of the reaction, SAH in cyan is localized near the catalytic site. The two Zn²⁺ ions (orange) on nsp10 are not involved in the interaction with nsp16.

Fig. 6

Sinefungin binding site and hypothetical RNA binding groove on SARS-CoV nsp16. Electrostatic potential surface of nsp16 presented with SAH (in cyan) and sinefungin (in yellow), which are localized in the same pocket. A putative binding groove for a capped-mRNA is indicated.

Fig. 7

Comparison of SARS-CoV nsp10 interaction surface with nsp14 and with nsp16. The SARS-CoV nsp10/nsp16 complex structure is presented with nsp10 solvent-accessible surface in gray, and nsp16 in yellow ribbon. Nsp10 residues involved in the interaction with nsp14 are depicted in green. The interaction surface of nsp10 with nsp16 and nsp14 overlaps, the latter being wider (Bouvet et al., 2014).