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Review
. 2020 May 20;9(5):1267.
doi: 10.3390/cells9051267.

A Structural View of SARS-CoV-2 RNA Replication Machinery: RNA Synthesis, Proofreading and Final Capping

Maria Romano  1 , Alessia Ruggiero  1 , Flavia Squeglia  1 , Giovanni Maga  2 , Rita Berisio  1
Affiliations
Review

A Structural View of SARS-CoV-2 RNA Replication Machinery: RNA Synthesis, Proofreading and Final Capping

Maria Romano et al. Cells. .

Abstract

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.

Keywords: COVID19; RNA replication; SARS-CoV-2; infectious disease; protein structure.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
SARS-CoV-2 19 polycistronic genome. (A) Genome of SARS-COV-2 organized in individual ORFs. (B) Polyprotein 1ab (PP1ab) embeds 16 non-structural proteins (Nsps); the black and grey triangles indicate the cleavage sites of the protease PLpro and 3CLpro, respectively. Names of confirmed and putative functional domains in the Nsps are also indicated.
Figure 2
Figure 2
Nsp12 and Nsp8 are a hub for interactions among actors of the RNA replication machinery. Predicted pattern of interactions based on the available literature data. The numbers 1 and 2 on the strings refer to SARS-CoV and MHV, respectively.
Figure 3
Figure 3
SARS-CoV-2 Nsp12-Nsp7-8 complex. (A) Domain organization of Nsp12, with its Nsp7 and Nsp8 cofactors, according to Pfam. (B) Cartoon representation of Nsp12 SARS-CoV-2 bound to Nsp7 and Nsp8 cofactors (PDB code 7BTF). (C) Model of SARS-CoV-2 elongation complex. The positions of the RNA template/primer and of the divalent cations were obtained from the structural alignment of the complex in panel A with the elongation complex from SARS-CoV-2 (PDB code 7BV2), while the position of NTP was obtained from the alignment with the polymerase of norovirus (PDB code 3H5Y). The three subdomains of the polymerase domain, finger (residues 366–581 and 621–679), palm (residues 582–620 and 680–815), and the thumb (residues 816–920) are shown in red, salmon and brown, respectively. (D) A zoom of the catalytic site showing the position of the incoming NTP, Remdesivir monophosphate (RMP) (in stick) and divalent cations (as green spheres). The conserved Asp residues that play a key role in the NTP and Mg2+ binding and Val557 (involved in Remdesivir resistance) are shown as yellow sticks.
Figure 4
Figure 4
Chemical structure of Remdesivir (GS-5734) and its pharmacologically active nucleoside triphosphate NTP.
Figure 5
Figure 5
Homology model of SARS-CoV-2 Nsp14-Nsp10 complex. (A) Domain organization of Nsp14 and Nsp10; (B) Cartoon representation of the homology model of the complex, computed with MODELLER using the structure of its homolog from SARS-CoV as a template (PDB code 5C8S, covered region 1−131 in Nsp10 and 1−525 in Nsp14). Zinc atoms are shown as grey and a Mg2+ ion as magenta spheres. Zooms of the catalytic sites are shown in the insets. Catalytic residues of the ExoN domain (left inset) are shown as blue sticks, those of the N7-MTase domain (right inset) are shown as orange sticks; the cap-precursor GpppA (pink), a SAH (demethylated form of SAM) ligand (yellow) and the SAM-binding motif residues (orange) are also represented as sticks.
Figure 6
Figure 6
The mRNA cap synthesis process in SARS-CoV-2. The process is performed by the sequential action of four enzymes: Nsp13 (pink), a still unknown GTase, Nsp14 (red) and Nsp16 (orange). The presence of the co-factor Nsp10 (grey) is fundamental for the activity of the last two enzymes.
Figure 7
Figure 7
SARS-CoV-2 Nsp13 helicase. (A) Domain organization of SARS-CoV-2 Nsp13. (B) Cartoon representation of the homology model of SARS-CoV-2 Nsp13, obtained using MODELLER based on the crystallographic structure of the SARS-CoV (PDB code 6JYT, covered region 1-596). The colors of the protein domains are indicated in panel A (ZBD-green, stalk-yellow, 1B-orange, 1A-red and 2A-salmon). Three zinc atoms are shown as grey spheres. In the inset, the key conserved residues responsible for NTP hydrolysis are drawn as sticks.
Figure 8
Figure 8
Structure of the SARS-CoV-2 Nsp10-Nsp16 complex. Cartoon representation of the crystal structure complex (PDB code 6W4H) of Nsp16 (orange) in complex with Nsp10 (grey). The inset shows the catalytic site of Nsp16. Catalytic residues (green) and the SAM ligand (yellow) are shown in stick form.
Figure 9
Figure 9
Nsp10 as a molecular connector between proofreading and capping activities. Cartoon and surface representation of the macromolecular complex generated upon superposition of Nsp10 in the two complexes Nsp14-Nsp10 and Nsp16-Nsp10. This model was obtained upon superposition of Nsp10 cofactors of the Nsp10/Nsp14 and Nsp10/Nsp16 complexes, using CCP4. A nascent RNA strand polymerized by Nsp12 (grey oval) is proofread by Nsp14, dephosphorylated by Nsp13 and then capped by GTase, Nsp14 and finally Nsp16.
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