SARS RNA Synthesis and Processing Activities – SARS-RNA-SPA

The expression of several viral enzymes from synthetic genes allows purifying multi-enzymatic complex. We are studying the regulation of viral enzymatic activities by biochemical methods and trying to crystallize proteins alone or in complex with their substrate to solve their structure. The catalytic residues are identified by site-directed mutagenesis and reverse genetics experiments and we demonstrate the key role of these enzymes in viral replication.

We have identified and characterized two partners of the polymerase essential for viral replication. We have characterized a processivity factor essential for genome replication and an exonuclease excising replication errors to ensure the genetic stability of the virus. Enzymatic essay were developed in order to identify polymerase inhibitors.

The development of an enzymatic assay to study the SARS-CoV polymerase should allow the identification of specific inhibitors. We will also determine how nsp14 exonuclease limits the action of inhibitors. We also hope to obtain the structure of the RNA polymerase in the presence and its processivity factor.

A paper is in preparation. We describe the mechanisms regulating nsp14 exonuclease activity by its nsp10 cofactor.

In 2003, a previously unknown coronavirus emerged from China and spread to the whole world, provoking more than 8000 cases of Severe Acute Respiratory Syndromes with a 10% case-fatality rate. Coronaviruses have the largest viral RNA genome known so far and exhibit a remarkable genetic stability. The replication machinery involves an enzyme set that is highly original and significantly more complex than that of most other RNA viruses. The replication/transcription complex carries I) RNA polymerase and RNA primase activities allowing the synthesis of genomic RNA and several 3’ nested subgenomic mRNA, II) a set of RNA modifying enzymes presumably involved in RNA capping such as RNA helicase/triphophatase and RNA N7-guanine and 2’O-methyltransferases, and III) RNA processing activities unique to Coronaviridae. Amongst these activities, nsp14 is a bifunctional RNA N7-guanine methyltransferase and RNA exonuclease, which could be involved in an RNA proofreading mechanism accounting for the observed genetic stability. The presence of nsp14 gene sequences in viral genomes is strictly correlated to unusually large RNA coronavirus genomes (>22 kb), since the closely related arteriviruses having RNA genome sizes <20 kb do not possess such enzyme. Nsp14 associates tightly to the replicative RNA polymerase nsp12, as well as with nsp10, an essential activator of the RNA-cap dependent 2’O-MTase.
Therefore, nsp14 is a rosette stone to decipher how the replication/transcription complex is structurally and functionally organized to perform, during viral replication, a wide variety of enzyme activities: allowing RNA capping and escape from host innate immunity, and mutation avoidance through potential RNA mismatch repair, a unique and yet unreported pathway in the RNA virus world.
We propose in this research program to address the following specific questions:
1. How the primase/polymerase pair ensures a highly processive synthesis of genomic and subgenomic RNA?
2. How the bifunctional nsp14 exonuclease/N7-methyltransferase mediates the proofreading activity during RNA polymerization?
3. How the viral cap structure synthesis is made and integrated in the replication process?
We will use a multidisplinary approach combining biochemical, viral and structural analysis to explore the fundamental processes of genetic stability and the interplay of RNA capping with innate immunity. These processes relate not only to antiviral drug and vaccine fields, but also to the larger field of RNA virus evolution and epidemiology of neglected emerging viral pathogens.