Identification and Characterization of a Ribose 2′- O -Methyltransferase Encoded by the Ronivirus Branch of Nidovirales

Abstract

The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae. RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2=-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2=-O-MTase activity, although we could not detect 2=-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2=-O-MTase, the roniviral 2=-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2=-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2=-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2=-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family. IMPORTANCEMethylation of the 5=-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2=-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales. In this study, we identified a 2=-Omethyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2=-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2=-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

Description and initial characterization of metatranscriptomic nidovirus-like genomes from the proposed new family Abyssoviridae, and from a sister group to the Coronavirinae, the proposed genus Alphaletovirus

“…The corresponding regions of AAbV and the top protein BLAST match were then submitted to HHPred in align mode, which uses predicted structure and primary sequence data to compare proteins. This led to confident identifications of the NiRAN and a match for the divergent but functional 2O MTase domain of Gill-associated virus (Zeng et al, 2016). One other uncharacterized domain was also identified in both AAbV and TurrNV by protein BLAST, in the position where the coronavirus conserved replication accessory proteins nsp7-10 were expected ( Fig.…”

Discrimination Between Self and Non-Self-Nucleic Acids by the Innate Immune System

“…The importance of viral RNA modifications in replication has been supported by studies that the infection of viruses lacking 2 0 -O-methyltransferase activity induces higher type I IFN expression, and unmodified genomic RNA was recognized by the host RNA sensor, MDA5 (Zust et al, 2011). The functional importance of 2 0 -O-methyltransferase activity for the infectivity of RNA viruses was also verified by studies on dengue virus (DENV) (Zust et al, 2018), Middle East respiratory syndrome coronavirus (MERS-CoV) (Aouadi et al, 2017), and ronivirus (Zeng et al, 2016). These viruses also encode 2 0 -O-methyltransferase and its disruption also causes type I IFN induction and virus elimination from the host following infection.…”

The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing

“…Although the (presumed) capping machinery of arteriviruses has remained essentially uncharacterized thus far (Lehmann et al, 2015b), three conserved putative capping enzymes were identified in the conserved ORF1b-encoded part of the replicase of Coronaviridae, Roniviridae, and Mesoniviridae, which all have substantially larger genomes. These enzymatic activities, which were proposed to participate in the synthesis of a cap-1 structure ( N7m GpppN 2 0 Om ), are the following: (i) the nsp13 helicase/RTPase (Ivanov and Ziebuhr, 2004), (ii) the nsp14 N7-MTase (Chen et al, 2009c;Ma et al, 2015), and (iii) the nsp16 2 0 -O-MTase (Bouvet et al, 2010;Decroly et al, 2008;Snijder et al, 2003;von Grotthuss et al, 2003;Zeng et al, 2016).…”