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| - RNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication (albeit within a permissive environment). A 33.5-kb nidovirus has been considered close to the upper size limit for such entities; conversely, the minimal cellular DNA genome is ~200 kb. This large difference presents a daunting gap for the transition from primordial RNP to contemporary DNA-RNP-based life. Whether or not RNA viruses represent transitional steps on the road to DNA-based life, studies of larger RNA viruses advance our understanding of size constraints on RNP entities. For example, emergence of the largest previously known RNA genomes (20-34 kb in positive-stranded nidoviruses, including coronaviruses) is associated with a proofreading exoribonuclease encoded in the nidoviral open reading frame 1b (ORF1b). However, apparent constraints on the size of ORF1b, which encodes this and other key replicative enzymes, have been hypothesized to limit further expansion of viral RNA genomes. Here, we characterize a novel nidovirus (planarian secretory cell nidovirus; PSCNV) whose disproportionately large ORF1b-like region, and overall 41.1 kb genome, substantially extend the presumed limits on RNA genome size. This genome encodes a predicted 13,556-aa polyprotein in an unconventional single ORF, yet retains canonical nidoviral genome organization and expression, and key replicative domains. Our evolutionary analysis suggests that PSCNV diverged early from multi-ORF nidoviruses, and subsequently acquired additional genes, including those typical of large DNA viruses or hosts. PSCNV’s greatly expanded genome, proteomic complexity, and unique features – impressive in themselves – attest to the likelihood of still-larger RNA genomes awaiting discovery. RNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication. The upper genome size for such entities was assumed to be <35 kb; conversely, the minimal cellular DNA genome is ~200 kb. This large difference presents a daunting gap for the proposed evolution of contemporary DNA-RNP-based life from primordial RNP entities. Here, we describe a nidovirus from planarians, whose 41.1 kb genome is 23% larger than the largest known of RNA virus. The planarian secretory cell nidovirus has broken apparent constraints on the size of the genomic subregion that encodes core replication machinery, and has acquired genes not previously observed in RNA viruses. This virus challenges and advances our understanding of the limits to RNA genome size.
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