Human SARS‐coronavirus RNA‐dependent RNA polymerase: Activity determinants and nucleoside analogue inhibitors

A coronavirus, an RNA virus, has been identified in humans as the pathogen responsible for the recent worldwide Severe Acute Respiratory Syndrome (SARS) outbreak. Rapid study of the critical SARS enzymes will provide useful inhibitors for this disease as well as a methodology that may be applicable to fight emerging pathogens in the future. The necessity of this rapid approach is further justified by the fact that the SARS virus reservoir has not been identified and future viral outbreaks are still possible. Indeed, new cases have already been reported in 2004. The SARS coronavirus (SARS CoV) genome is a positive strand of RNA with 11 open reading frames and a genomic organization similar to other coronaviruses. However, phylogenic analysis and sequence alignments have shown that the SARS CoV has developed distinctly from other coronaviruses. The viral replication proceeds via synthesis of a complementary minus strand RNA using the genome as a template and the subsequent synthesis of genomic plus strand RNA from the minus strand RNA template. The key enzyme responsible for both steps is the RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 12 (nsP12). Since RdRp activity is not essential for the host, its inhibition will not cause undesirable side effects during therapy. With the enzyme characterization in its early stages, a homology model based on the amino acid sequence can provide some clues concerning the residues critical for its function. We have built a homology model of the SARS polymerase using the 3D jury system. Recently, during preparation and revision of this study, Xu et al. reported an RdRp model using the program Modeller. Although their and our models have been built using a different approach, the r.m.s between the two models is about 1.6 Å (for C atoms). The similarity between the two models can be partially explained by the fact that the 3D-jury system uses a set of prediction methods that includes the Pcons method on which the Modeller program is based on. The capability of the 3D-jury in using consensus between Meta prediction methods makes it a very reliable tool for model building as proven by its highest ranking in the CASP5 (Critical Assessment of Techniques for Protein Structure Prediction) competition. This predictor has been used recently to successfully assign a methyl-transferase function to nsP13 and to build a 3D model that identified residues critical for the enzyme function. Substrate and ligand docking in the active site were carried out with the Genetic Optimization Ligand Docking Program (Gold) giving us an estimate of ligand binding in the active site. This docking method, with a success rate of around 70% through its validation process, proved to be very reliable when used with caution.