ABSTRACT
Nidovirales is an extraordinary order of complex positive-stranded RNA viruses including some of the largest RNA genomes (12-41 kb) among which notable human health burdens: SARS-CoV-1, SARS-CoV-2, MERS-CoV, etc. Recent advance in genome sequencing is slowly filling the gaps between and beyond the classified nidoviral families. Still, the research is lagging behind to understand the evolution of RNA genomes. For example, how are these large genome RNA viruses able to bypass the length and stability constraints of an RNA molecule? Is there any link between increasing length and gaining a functional domain or a special structural feature? To answer these questions, we started with database mining to extract novel nidoviral genomes and annotated different domains in polyproteins of classified and unclassified nidoviruses using HHpred and HHblits tools (Zimmermann L, et al. 2018). We observed a significant variation across the order regarding presence/absence, fold/structure type, co-factor (or enhancer) presence/absence, presence of one motif or the other and genome location of enzymes: Exonuclease (ExoN), N-7 Methyltransferase (MTase), 2'-O-MTase and RNA dependent RNA polymerase (RdRp). A trend seen with this bioinformatic analysis directly implies that stable RNA genome increase as well as maintenance is driven by the synergy of modifying enzymes: MTases, RNA proofreading by ExoNs and fast & processive RdRps (Ferron F, et al. 2021). Next, after their identification, we are trying to characterize these large RNA genome genetic markers: MTase(s) & ExoN, to have a comprehensive understanding of nidoviruses evolution. We have identified, expressed and purified a new nidoviral MTase from a Tobaniviridae family member, White Bream Virus (WBV). This enzyme is unique in terms of its location in ORF1a and not in ORF1b (Ferron F, et al. 2019). Functional and mutational studies show this new MTase to contain N-7 guanine specific, S-adenosyl-methionine (SAM) dependent capping activity (cap-0). Aligning with our predictions, structural characterization confirms that it has a Rossmann fold (RF) SAMdependent RNA-cap N7-guanine MTase. This study answers the missing link of capping activity in these members, which is somewhat only established for coronaviruses in this large genome order. Evaluating such enzymes is a step forward in the direction of fundamental understanding of how these RNA viruses are successfully expanding and maintaining their large genomes as well as coping up to fight against the host innate immunity.
ABSTRACT
With the current Covid-19 ongoing outbreak originating inWuhan, human pathogenic coronaviruses demonstrate their ability to emerge abruptly and spread serious pulmonary disease. The Orf1b enzymes promote replication and transcription within a complex with unique enzyme having outstanding properties. The RdRp core sequence of the Covid-19 isolate published in january 2020 shows a high (>95% aa) sequence homology to the SARSCoV emerged in 2003. Analysis of polymorphisms show that aa changes are mostly located at the protein surface, unlikely to affect any basic function of the RdRp.We have reconstituted a highly active SARS-CoV RdRp complex made of nsp7, nsp8, and nsp12, and studied its polymerization activity on a variety of RNA templates using steady-state and pre-steady state kinetics. The RdRp is able to incorporate single NTPs at the astonishing rate of >500 s-1, about 10-fold faster than any known viral RdRp. Fast synthesis occurs at the expense of fidelity, which is at least 10-fold lower than that of Dengue virus NS5 or Coxsackie virus RdRps. Such low fidelity must be corrected by the nsp14 ExoN subdomain -able to remove 3'-terminal mismatches to match genome stability observed in infected cells, and account for the large size of the Coronavirus RNA genome. The nsp14 enzyme is a bi-functional enzyme made of an Exonuclease domain, activated through binding to nsp10, and an RNA methyltransferase able to execute N7-guanine methylation of RNA caps. It is the only example of RNA cap MTase which does not have a Rossmann fold, which poses interesting questions given the overwhelming success of the Rossmann fold through evolution. Nsp13 is a type 1 helicase, whose role is unclear. The nsp15 RNA endonuclease is a RNase A type endonuclease specific for Uracile, inhibited by 2'-O methylation of RNA, while nsp16 is a 2'-O methyltransferase also activated by nsp10. Our work provides a structural and functional view of this sophisticated replication complex, with highly active enzyme preparations suitable for robotized high-throughput inhibition assays aiming at the discovery of pan-coronavirus inhibitors Orf1b enzymes.