Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.
Highlights
To capture the full coding capacity of SARS-CoV-2, we applied a range of ribosome-profiling approaches to Vero E6 cells infected with SARS-CoV-2 for 5 or 24 h (Fig. 1a)
Ribosome footprints displayed a strong peak at the translation initiation site, which, as expected, is more pronounced in the Harr and LTM libraries; the CHX library exhibited a distribution of ribosomes across the entire coding region, and the mapped ribosome footprints were enriched in fragments that align to the translated frame (Fig. 1b, Extended Data Fig. 2a)
The translation efficiencies of ORF1a and ORF1b were considerably lower. This may stem from distinct features in their 5′ untranslated region (UTR) or from under estimation of their true translation efficiency, as some of the full-length RNA molecules may serve as template for replication or packaging and are not part of the translated mRNA pool
Summary
Yaara Finkel[1,7], Orel Mizrahi[1,7], Aharon Nachshon[1], Shira Weingarten-Gabbay[2,3], David Morgenstern[4], Yfat Yahalom-Ronen[5], Hadas Tamir[5], Hagit Achdout[5], Dana Stein[6], Ofir Israeli[6], Adi Beth-Din[6], Sharon Melamed[5], Shay Weiss[5], Tomer Israely[5], Nir Paran[5], Michal Schwartz1 & Noam Stern-Ginossar1 ✉. To facilitate mapping of translation initiation sites, we prepared two Ribo-seq libraries by treating cells with lactimidomycin (LTM) or harringtonine (Harr), two drugs with distinct mechanisms that prevent elongation at 80S ribosomes at translation initiation sites. These treatments lead to excessive accumulation of ribosomes precisely at the sites of translation initiation and depletion of ribosomes over the body of the ORF (Fig. 1a). The footprint profiles of viral coding sequences at 5 hpi fit the expected profile of translated sequences (Fig. 1c, Extended Data Fig. 2b) and the footprint densities were highly reproducible between biological replicates, at single-nucleotide resolution
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