PNNL

Abstract

Coronaviruses (CoVs) are continually emerging and possess some of the largest RNA genomes in nature. Since 2003, three emerging CoVs capable of causing severe human disease have resulted in millions of deaths and billions in economic damages worldwide, underscoring the importance of defining the molecular determinants of replication and virulence, especially those involving the various viral RNA species produced during infection. During their replication cycles, CoVs reproduce genome-length RNAs and generate 3’-nested co-terminal sets of subgenomic RNAs (sgRNAs) that serve as mRNAs for the translation of downstream ORFs. The production of these various RNA species is mediated by a complex minus-strand discontinuous transcription program that involves double-stranded intermediate RNAs and the acquisition of a ~70-nt leader sequence from the genomic 5’ end. As the RNA biology of viruses has important implications for evolution, replication, gene expression and immune surveillance, we used multiple deep-sequencing approaches to characterize the viral plus- and minus-strand RNA transcriptome and proteome to reveal novel translation processes that occur during MERS-CoV infection. Intriguingly, our analyses revealed complex networks of subgenomic mRNA regulated by canonical and noncanonical transcription regulatory sequences, including multiple noncanonical leader- and antileader-containing RNA species, indicating that the RNA transcription program of MERS-CoV is far more complex than previously recognized. Moreover, viral protein production occurred from both canonical and noncanonical loci, suggesting that the proteomes of CoVs are considerably more complex, challenging current paradigms of coronavirus genome organization and function. This study greatly expands our understanding of the CoV transcriptome/proteome and offers multiple novel potential RNA and protein targets for modulating replication and virulence, potentially including early protogenes that may serve as substrates for adaptive evolution and the emergence of novel viral functions.