Fe-S clusters as essential cofactor of Coronavirus replication – CoV-FeS

Replicating and maintaining genome integrity is an upmost task for all organisms. To replicate their exceptionally large genome, Coronaviruses (CoVs) employ a unique and sophisticated RNA-synthesis and correction machinery, composed of, at least, the RNA-dependent RNA polymerase (RdRp, nsp12), the helicase (nsp13), the 3’-5’ exonuclease (ExoN, nsp14) and its cofactor nsp10, which ensures the RNA proofreading mechanism. While these three enzymatic activities are essential for genome stability maintenance and therefore must work in concert, little is known on how they communicate together, i.e., how ExoN recruitment takes place when a mismatch is inserted into the neosynthesized RNA by the replicase complex.
Recently, the finding that the SARS-CoV-2 RdRp (nsp12) and the helicase (nsp13) accommodate Fe-S clusters instead of zinc ions, as initially described, unveiled unsuspected aspects of the virus. Our preliminary data indicate that the 3’-5’ exonuclease (ExoN, nsp14) and its cofactor nsp10 might also bind Fe-S clusters. Altogether, this provides a strong example that virus can use Fe-S proteins to ensure a crucial function in their life cycle. Actually, the key role of Fe-S cluster in viral proteins is an emerging field, in which CoVs stand out as models. Also, this is a striking illustration of the importance of Fe-S proteins in replicating and maintaining genome integrity, a domain in which over time more and more Fe-S proteins are shown to be exploited.
By analogy with the critical role of Fe-S clusters in DNA repair mechanisms, we hypothesize that in the unique CoV RNA proofreading pathway, involving nsp12/nsp13/nsp14/nsp10 players, protein-protein dialogs amongst them are crucial and involve Fe-S clusters.
The CoV-FeS program aims to (1) investigate at the single-molecule scale the contribution of the Fe-S cluster for communication between SARS-CoV-2 RNA polymerase (nsp12) and helicase (nsp13), as well as with their RNA substrate, and (2) to establish further nsp14-ExoN and nsp10 as Fe-S proteins.
An unfortunate consequence of the use of Fe-S clusters is their vulnerability to oxidants, and their possible mis-metalation as illustrated by the fact that Fe-S proteins are often mischaracterized as zinc-containing proteins. However, in the case of our fight against SARS-CoV-2, this drawback might be turned in an advantage to pave the way for anti-CoV compounds. This will be the (3) and last aim of our project, which is to gain information on how the Fe-S CoV proteins acquire their clusters and what cellular conditions that might be unfavorable for the stability of their cluster.
To this end, the CoV-FeS project has gathered a multidisciplinary consortium that will deploy: (i) approaches to produce, characterize and identify Fe-S proteins; (ii) cutting-edge single-molecule assay with the development of trapping instrumentation to study the CoV RNA proofreading mechanism; and (iii) a panel of E. coli and yeast strains engineered to investigate how to impede Fe-S cluster stability and/or acquisition in CoV [FeS]-proteins
More broadly, this program will lay the groundwork for characterizing roles of viral Fe-S proteins and their clusters for optimal viral genome replication. Furthermore, the CoV-FeS program will provide a methodological pipeline for their studies. All this knowledge will guide the establishment of new strategies to counteract (re)-emergent viral infections.