RNA genome expansion up to 64 kb in nidoviruses: role of host, genome segmentation, translation elongation and replicase expression
Positive-strand RNA viruses of the order Nidovirales have the largest known RNA genomes of vertebrate and invertebrate viruses with 36.7 and 41.1 kb, respectively. The acquisition of a proofreading exoribonuclease (ExoN) locus by an ancestral nidovirus enabled crossing of the 20 kb barrier. Other factors constraining genome expansions in nidoviruses remain poorly defined. Here, we assemble 76 genome sequences of invertebrate nidoviruses from >500.000 published transcriptome experiments and triple the number of known nidoviruses with >36 kb genomes, including the largest known 64 kb RNA genome. Many of the novel viral lineages acquired putative enzymatic domains that were inserted in open reading frame (ORF) 1a and ORF1b or equivalent regions and may constitute cofactors of the viral replicase or modulate infection otherwise. We classify multi-cistronic ExoN-encoding nidoviruses into seven groups and four subgroups, according to canonical and non-canonical modes of viral polymerase expression by ribosomes and genomic organization (reModes). The largest group employing the canonical reMode comprises invertebrate and vertebrate nidoviruses, including coronaviruses, with genomes ranging from 20 to 36 kb. Six groups with non-canonical reModes include giant invertebrate nidoviruses with 31 to 64 kb genomes. Among them are viruses with segmented genomes and viruses utilizing dual ribosomal frameshifting that we validate experimentally. Moreover, polyprotein length and genome size in nidoviruses show reMode- and host phylum-dependent relationships. We demonstrate that the largest polyproteins in nidoviruses may be close to an upper limit that we hypothesize to be determined by the host-inherent translation fidelity, further constraining nidovirus genome size. Thus, expansion of giant RNA virus genomes, the vertebrate/invertebrate host division, the control of viral replicase expression, and translation fidelity are interconnected. Overall design: DMS-MaP analysis of in vitro transcribed RNA