Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design

Abstract

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV plays a pivotal role in viral replication and is a potential target for anti-SARS therapy. There is a lack of structural or biochemical data on any coronavirus polymerase. To provide insights into the structure and function of SARS-CoV RdRp, we have located its conserved motifs that are shared by all RdRps, and built a three-dimensional model of the catalytic domain. The structural model permits us to discuss the potential functional roles of the conserved motifs and residues in replication and their potential interactions with inhibitors of related enzymes. We predict important structural attributes of potential anti-SARS-CoV RdRp nucleotide analog inhibitors: hydrogen-bonding capability for the 2' and 3' groups of the sugar ring and C3' endo sugar puckering, and the absence of a hydrophobic binding pocket for non-nucleoside analog inhibitors similar to those observed in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase. We propose that the clinically observed resistance of SARS to ribavirin is probably due to perturbation of the conserved motif A that controls rNTP binding and fidelity of polymerization. Our results suggest that designing anti-SARS therapies can benefit from successful experiences in design of other antiviral drugs. This work should also provide guidance for future biochemical experiments.

Figure 1

Sequence alignment of the RdRp of SARS-CoV with those of representatives of the other three classes of coronaviruses and of five RNA viruses with known crystal structures. The representative coronaviruses are: group I, human coronavirus 229E (HcoV-229E; NCBI accession no. NC_002645); group II, murine hepatitis virus (MHV; NCBI accession no. NC_001846); and group III, avian infectious bronchitis virus (AIBV; NCBI accession no. NC_001451). The five RNA viruses are poliovirus 1 strain Mahoney (PV; PDB code 1RDR), rabbit hemorrhagic disease virus (RHDV; PDB code 1KHV), hepatitis C virus (HCV; PDB code 1QUV), reovirus (RV; PDB codes 1N35 and 1N1H) and bacteriophage φ6 (Phi6; PDB codes 1HI0 and 1HI1). HIV-1 RT (HIV-1; PDB code 1RTD), a widely studied RNA-dependent and DNA-dependent polymerase, is also included in the comparison. The sequence in the palm subdomain and regions containing the conserved motifs (highlighted with green bars) can be aligned confidently among different viral RdRps and HIV-1 RT. However, the sequence in the fingers and thumb subdomains is less conserved between SARS-CoV RdRp and other viral RdRps, and the structure in those subdomains also varies substantially among the known RdRp structures. Thus, the sequence alignment and the structural model in these regions are less reliable. Invariant residues are highlighted in a shaded red box, and conserved residues are in red. The secondary structures of RHDV, HCV, RV and φ6 polymerases extracted from the corresponding structures and the predicted secondary structure of SARS-CoV RdRp are shown above the sequence alignment. α-Helices are shown as spirals and β-strands as arrows. The alignment was drawn with ESPript (66).

Figure 1

Sequence alignment of the RdRp of SARS-CoV with those of representatives of the other three classes of coronaviruses and of five RNA viruses with known crystal structures. The representative coronaviruses are: group I, human coronavirus 229E (HcoV-229E; NCBI accession no. NC_002645); group II, murine hepatitis virus (MHV; NCBI accession no. NC_001846); and group III, avian infectious bronchitis virus (AIBV; NCBI accession no. NC_001451). The five RNA viruses are poliovirus 1 strain Mahoney (PV; PDB code 1RDR), rabbit hemorrhagic disease virus (RHDV; PDB code 1KHV), hepatitis C virus (HCV; PDB code 1QUV), reovirus (RV; PDB codes 1N35 and 1N1H) and bacteriophage φ6 (Phi6; PDB codes 1HI0 and 1HI1). HIV-1 RT (HIV-1; PDB code 1RTD), a widely studied RNA-dependent and DNA-dependent polymerase, is also included in the comparison. The sequence in the palm subdomain and regions containing the conserved motifs (highlighted with green bars) can be aligned confidently among different viral RdRps and HIV-1 RT. However, the sequence in the fingers and thumb subdomains is less conserved between SARS-CoV RdRp and other viral RdRps, and the structure in those subdomains also varies substantially among the known RdRp structures. Thus, the sequence alignment and the structural model in these regions are less reliable. Invariant residues are highlighted in a shaded red box, and conserved residues are in red. The secondary structures of RHDV, HCV, RV and φ6 polymerases extracted from the corresponding structures and the predicted secondary structure of SARS-CoV RdRp are shown above the sequence alignment. α-Helices are shown as spirals and β-strands as arrows. The alignment was drawn with ESPript (66).

Figure 2

Ribbon diagram of the homology model of SARS-CoV RdRp with a docked RNA template–primer. α-Helices are shown as spirals and β-strands as arrows. The subdomains of the catalytic domain are colored as the N-terminal portion of the fingers subdomain (376–424) in magenta, the base of the fingers (residues 425–584 and 626–679) in blue, palm (residues 585–625 and 680–807) in red, and thumb (residues 808–932) in green.

Figure 3

Stereoview of the polymerase active site and the rNTP-binding site. The conserved sequence motifs (A–G) are highlighted. A docked rNTP substrate is shown as a ball-and-stick model. The catalytic active site is defined by the three conserved aspartates, Asp618, Asp760 and Asp761 (shown with side chains) that are coordinated with two divalent metal ions (shown as magenta spheres).

Figure 4

Structural comparison of HCV, PV, RHDV, RV, φ6 and SARS-CoV RdRps and HIV-1 RT in the regions containing motifs D and E. (A) Ribbon presentation of motifs D and E in the structures of HCV, PV, RHDV, RV, φ6 and HIV-1 polymerases, and in the structural model of SARS-CoV polymerase. (B) Superposition of the regions containing motifs D and E in different viral RdRp and RT structures [the color coding is the same as in (A)].

Figure 4

Structural comparison of HCV, PV, RHDV, RV, φ6 and SARS-CoV RdRps and HIV-1 RT in the regions containing motifs D and E. (A) Ribbon presentation of motifs D and E in the structures of HCV, PV, RHDV, RV, φ6 and HIV-1 polymerases, and in the structural model of SARS-CoV polymerase. (B) Superposition of the regions containing motifs D and E in different viral RdRp and RT structures [the color coding is the same as in (A)].