Discovery of the order ‘ Quisvirales ’ redefines the evolution of RNA replication and transcription in the phylum Pisuviricota

Abstract

Genome replication in positive-stranded RNA (ssRNA+) viruses is mediated by cognate enzymes, including ubiquitous RNA-dependent RNA polymerase (RdRp). In ssRNA+ viruses with multiple open reading frames (ORFs) in their genomes, replication is accompanied by synthesis of subgenomic RNAs (transcription) for expression of 3′-proximal ORFs. In addition, all ssRNA+ viruses with genomes larger than ~ 7 kb encode helicases, linking helicases to RNA genome expansion. Helicases are essential ATPases that unwind nucleic acids and are classified into six recognized superfamilies (SF1–SF6). In the phylum Pisuviricota that includes important pathogens, helicases of SF1-SF3 are integrated into multi-enzyme replicase polyprotein(s) including 3C(−like) protease (3CLpro) and RdRp. Here, large-scale mining of invertebrate metatranscriptomes and targeted genome sequence assembly uncovered six spider-associated ssRNA+ viruses that, based on their conserved 3CLpro–RdRp module in replicase polyproteins, genome size (20–22 kb), and phylogeny, form a family-like cluster in a putative order, named ‘Quisvirales’. Quisviruses have similar genome and replicase architectures to enveloped coronaviruses and other nidoviruses. Notably, quisviruses encode ORFs 1a and 1b with predicted −1 programmed ribosomal frameshifting elements in the ORF1a/b overlap region. Using an original mapping approach for detecting chimeric sequencing reads, we obtained evidence that 3′-proximal ORFs are expressed via 5′-coterminal, leader-containing subgenomic RNAs. This suggests that the quisvirus subgenomic RNAs are generated through discontinuous transcription—a mechanism otherwise exclusively found in nidoviruses among the many ssRNA+ virus orders that synthesize subgenomic RNAs. A striking difference between nido- and quisviruses is, however, the RdRp being the only common core ORF1b-encoded enzyme and the replacement of the nidovirus SF1 helicase by a novel superfamily helicase. This quisvirus SF7 helicase, like the Picornavirales SF3 helicase, comprises an AAA+ (ATPase-like) domain typical for ring-forming helicases and thus must play an essential role in replication. The discovery of the order ‘Quisvirales’ demonstrates that viruses employing large replicase polyproteins of nidovirus-like complexity and discontinuous transcription may have evolved repeatedly from an 3CLpro–RdRp-encoding ancestor.