Replication of positive stranded RNA viruses in virus-infected cells is believed to be catalyzed by viral replicase complexes, which may consist of various virally encoded nonstructural proteins including the RNA dependent RNA polymerases (RdRp), and host factors. The multi-domain replicase gene 1 of the SARS coronavirus (SARS-CoV), the recently identified causative agent of Severe Acute Respiratory Syndrome (SARS) encompasses nearly two-thirds of its genome (approximately 22 kb) and is predicted to encode two overlapping polyproteins, ppla (replicase la) and pplab (replicase lab), which undergo cotranslational proteolytic processing to yield a number of functional polypeptides required for viral replication and transcription. Non-structural protein 9 (nsp9), a putative proteolytic product of replicase 1ab is proposed to encode the putative viral RNA dependent RNA polymerase (RdRp) or POL domain, the protein responsible for synthesizing the viral positive strand RNA genome via (-) strand intermediate and therefore represents an attractive target for therapeutic intervention. The goal of this application is to identify inhibitors of this protein. To achieve this, we propose to generate a functionally active SARS-CoV RdRp and establish its biochemical characteristics pertaining to RNA synthesis, substrate utilization and other enzymatic attributes via analogy to other members of the RNA dependent RNA polymerase family. Given the complexity of the replicase gene and the lack of enzymatic and biochemical data on any Coronavirus RdRp, our attempt to generate a functionally active SARS-CoV polymerase capable of replicating the entire 30 kb viral genome remains highly exploratory in nature. We propose to clone the putative polymerase domain (nsp9) of the SARS-CoV in both prokaryotic and eukaryotic expression vectors (in case it requires post-translational modification) and purify the active protein. Another important aspect of these studies is to identify the essential structural elements and domains required to generate a functionally active polymerase. Towards this objective, we have identified a "conserved RdRP domain" of 560 amino acids spanning residues 373 to 932 in the putative SARS-POL domain including the characteristic XDD signature motif of RdRp. Based on secondary structure predictions, we will construct three N-terminal truncation clones of SARS-CoV RdRp, and delineate the contribution of the alpha-helices and beta-sheets formed by the initial N terminal 372 residues. SARS-POL domain contains five "XDD motifs". We propose to experimentally elucidate its catalytic "XDD motif". This will establish similarities and differences between the SARS-CoV RdRp versus all other coronavirus RdRps. To establish the enzymatic activity of the various constructs generated by this approach, specific conditions such as those used by hepatitis C virus replicase, poliovirus replicase, HIV-1 RT and other related RdRp will be utilized. It is possible that SARS-CoV RdRp may exhibit complete variance in its enzymatic characteristic that will also be examined. The proposed investigation will thus establish a new knowledge base for the SARSCoV RdRp in particular and members of the Coronoviridiae RdRp family in general. Further, analysis of the sensitivity of the SARS-CoV RdRp towards a panel of known and novel inhibitors of polymerases will provide new leads in the quest for specific antiviral agents against the SARS-CoV RdRp.