Subgenomic messenger RNA amplification in coronaviruses

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Abstract
Coronaviruses possess the largest known RNA genome, a 27- to 32-kb (+)-strand molecule that replicates in the cytoplasm. During virus replication, a 3' coterminal nested set of five to eight subgenomic (sg) mRNAs are made that are also 5' coterminal with the genome, because they carry the genomic leader as the result of discontinuous transcription at intergenic donor signals during (-)-strand synthesis when templates for sgmRNA synthesis are made. An unanswered question is whether the sgmRNAs, which appear rapidly and abundantly, undergo posttranscriptional amplification. Here, using RT-PCR and sequence analyses of head-to-tail-ligated (-) strands, we show that after transfection of an in vitro-generated marked sgmRNA into virus-infected cells, the sgmRNA, like the genome, can function as a template for (-)-strand synthesis. Furthermore, when the transfected sgmRNA contains an internally placed RNA-dependent RNA polymerase template-switching donor signal, discontinuous transcription occurs at this site, and a shorter, 3' terminally nested leader-containing sgmRNA is made, as evidenced by its leader-body junction and by the expression of a GFP gene. Thus, in principle, the longer-nested sgmRNAs in a natural infection, all of which contain potential internal template-switching donor signals, can function to increase the number of the shorter 3'-nested sgmRNAs. One predicted advantage of this behavior for coronavirus survivability is an increased chance of maintaining genome fitness in the 3' one-third of the genome via a homologous recombination between the (now independently abundant) WT sgmRNA and a defective genome.