Coronavirus RNA synthesis: how to handle errors, stops and jumps while replicating the largest RNA genome.

Due to the intrinsic high error frequency of their replicative machinery, the apparent absence of proofreading or repair mechanisms, and their explosive replication, the rate of positive-strand (+RNA) virus evolution is unparalleled. Although their error-prone replication (estimated mutation frequency 10-4) has clearly limited the genome size of +RNA viruses, the diverse repertoire of evolutionary ?tools? is a major factor in the ceaseless arms race between virus and host and in the emergence of novel virus infections. Understanding the molecular details of viral RNA synthesis will be crucial for the development of novel and universal strategies to combat RNA viruses. Among +RNA viruses, coronaviruses stand out for their unusually large genome size of 27-31 kilobases, which is about 3-fold larger than the average +RNA virus genome size. The coronavirus replicase/transcriptase is an extremely complex ~7000 amino acid protein that is autoproteolytically cleaved into 16 nonstructural proteins (nsp1-16), which are assumed to be the active enzymes in viral RNA synthesis. Among these are key enzymes for viral RNA synthesis, like the RNA-dependent RNA polymerase (RdRp; nsp12) and helicase (nsp13) that are found in other +RNA viruses, but also many poorly defined proteins that appear to be unique for coronaviruses and their closest relatives. Intensified structural and bioinformatics studies, which were triggered by the recent emergence of SARS-Coronavirus (SARS-CoV), have now implicated many of those proteins in viral RNA synthesis. Among the newly discovered functions are several RNA-binding proteins (nsp7-8-9, with nsp7 and nsp8 forming a unique hexadecameric hollow structure that can encircle double-stranded RNA), a 3?-to-5? exonuclease (nsp14) and an endoribonuclease (nsp15). These proteins were implicated in RNA metabolism and in particular in reducing/compensating RdRp infidelity, ensuring RdRp processivity, and/or regulating the balance between genome replication and the unique mechanism for subgenomic mRNA production that operates in coronaviruses. The emergence of SARS-CoV in 2003 has intensified our contacts with a number of foreign laboratories. In particular in the area of structural genomics of the coronavirus replicase, our group is now engaged in a productive collaboration with the groups of Dr. Bruno Canard, Dr. Christian Cambillau, and Dr. Jean-Claude Guillemot in Marseille, France, who started a genome-wide approach to express, purify, and crystallize coronavirus replicase subunits. Additional funding is now required to continue this collaboration on the structure, biochemistry, and molecular biology of selected coronavirus targets (nsp7-8-9, nsp14, and nsp15) for SARS-CoV and the best studied animal coronavirus model, Mouse hepatitis virus (MHV). Structural studies will be performed in Marseille, the biochemistry will be done in collaboration between the two labs, and the function of the proteins in viral RNA synthesis and evolution will be addressed in Leiden, where extensive coronavirus experience, all necessary reagents, and the appropriate containment facilities are available. The central hypothesis of this project is that coronaviruses have evolved a specialized set of accessory protein functions to assist the RdRp in coping with the specific problems of amplifying a large RNA genome and regulating the complex process of genomic and subgenomic RNA synthesis. Using the currently available and anticipated structural data, the project aims to dissect the biochemistry of these accessory proteins and assess their role in virus replication and evolution. Various questions that are directly related to the fidelity, processivity, and bifunctionality (replication vs. transcription) of coronavirus RNA synthesis will be addressed: · Which coronavirus replicase subunits are (functionally) associated with the nsp12 ?core RdRp? ? · Does coronavirus replication rely on a mechanism compensating the high error frequency of the RdRp complex? · Does the RdRp complex contain specific factors that stimulate its processivity while copying a ~30 kb template? · Is the balance between continuous and discontinuous RNA synthesis (genome replication versus subgenomic RNA production) regulated by specific replicase subunits? Our technical approach is based on using in vitro assays for defining interactions between coronavirus nsps. Also interactions between nsps and general or virus-specific RNA sequences will be addressed. Structure-based mutagenesis will be an important tool, which can be extended to the complete virus life cycle using the available systems for coronavirus reverse genetics. This will also allow the identification of interaction partners on the basis of second-site revertants and enable us to study the phenotype and evolution of mutants that are impaired (but not fully blocked) in specific nsp functions.