Canard, B. | bioRxiv Science

bioRxiv2019

Identification of a Nidovirales Orf1a N7-guanine cap methyltransferase signature-sequence as a genetic marker of large RNA genome Tobaniviridae

Members of the Nidovirales order have (+)RNA genomes amongst the largest in size in the RNA virus world. Expression of their genes is promoted through reading of genomic RNA and mRNA transcripts by the ribosome of the infected cell. The 5-end of these RNAs is supposedly protected by an RNA-cap structure (m7GpppNm) whose most synthesis steps remain elusive. In Eukaryotes, the RNA-cap structure is methylated by RNA methyltransferases (MTases) at the RNA-cap N7-guanine position as well as the 2-O methyl position of the first transcribed nucleotide. In Coronaviridae, two separate enzymes (nsp14 and nsp16) perform the N7-guanine and the 2-OH methylation, respectively. One salient feature of the Nidovirales N7-guanine MTase nsp14 is that it is the only example of non-Rossman fold viral MTase known so far. Conversely, all other Nidovirales nsp16-like MTases have a canonical Rossman fold. Many Nidovirales members lack either any RNA MTase signature sequence (e.g., Arteriviridae), or lack a N7-guanine MTase signature sequence (e.g., Tobaniviridae, Euroniviridae, Roniviridae, Medioniviridae). Both nsp14-and nsp16-like enzyme genes are usually located in Orf1b encoding for the replication machinery. Here, we report the discovery of a putative Rossman fold RNA MTase in the Orf1a of ten Tobaniviridae members. Multiple sequence alignments and structural analyses identify this novel gene as a typical RNA-cap N7-guanine MTase with substrate specificity and active-site organization similar to the canonical eukaryotic RNA-cap N7-guanine MTase.

bioinformatics↗

bioRxiv2019

Arenaviridae exoribonuclease presents genomic RNA edition capacity.

The Arenaviridae is a large family of viruses causing both acute and persistent infections and causing significant public health concerns in afflicted regions. A "trademark" of infection is the quick and efficient immuno-suppression mediated in part by a 3-5 RNA exonuclease domain (ExoN) of the Nucleoprotein (NP). Mopeia virus, the eastern African counterpart of Lassa virus, carries such ExoN domain, but does not suppress the host innate immunity. We have recently reported the crystal structure of the Mopeia virus ExoN domain, which presents a conserved fold and active site. In the present study, we show that the ExoN activity rules out a direct link between ExoN activity and alteration of the host innate immunity. We found that the Arenavirus ExoN, however, is able to excise mis-incorporated bases present at the 3-end of double stranded RNA. ExoN(-) arenaviruses cultured in cells dampened in innate immunity still replicated in spite of a significant reduction in the viral charge over several passages. The remaining ExoN(-) virus population showed an increased base substitution rate on a narrow nucleotide spectrum, linking the ExoN activity to genome editing. Since, the Arenavirus ExoN belongs to the same nuclease family as that of the nsp14 coronavirus ExoN ; which has been recently shown to promote viral RNA synthesis proofreading; we propose that Arenavirus ExoN is involved in a "limited RNA editing" mechanism mainly controlled by structural constraints and a low mutational/fitness ratio. Author summaryOnly Arenaviridae and Coronaviridae encode a 3-5 RNA exonuclease domain (ExoN) in their genome. This activity is either used to counteract the innate immunity response during viral infection or to ensure genome stability during replication. Mopeia virus (MOPV), the eastern African counterpart of Lassa virus, carries such ExoN domain, but does not suppress the host innate immunity. We studied MOPV ExoN activity both in vitro and in cellula to assess the role of ExoN MOPV and found that the Arenaviral ExoN is fully active on dsRNA, and is able like the one of Coronaviridae to excise a mismatched base. We measured genetic stability and found evidence of a limited spectrum of RNA synthesis proofreading mechanism, together with a strongly impacted viral replication. We propose that the Arenaviral ExoN is involved in a functional check of the conserved RNA structures of the viral genome.

biochemistry↗