SARS-CoV-2 utilizes a multipronged strategy to suppress host protein synthesis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. Here, we use RNA-sequencing and ribosome profiling along SARS-CoV-2 infection and comprehensively define the mechanisms that are utilized by SARS-CoV-2 to shutoff cellular protein synthesis. We show SARS-CoV-2 infection leads to a global reduction in translation but that viral transcripts are not preferentially translated. Instead, we reveal that infection leads to accelerated degradation of cytosolic cellular mRNAs which facilitates viral takeover of the mRNA pool in infected cells. Moreover, we show that the translation of transcripts whose expression is induced in response to infection, including innate immune genes, is impaired, implying infection prevents newly transcribed cellular mRNAs from accessing the ribosomes. Overall, our results uncover the multipronged strategy employed by SARS-CoV-2 to commandeer the translation machinery and to suppress host defenses. To gain a detailed view of the changes that occur in viral and host translation over the course of SARS-CoV-2 infection, we applied ribosome profiling and RNA-sequencing to Calu3 cells infected with SARS-CoV-2 as well as uninfected cells. The cells were infected with SARS-CoV-2 BavPat1/2020 Ref-SKU: 026V-03883 at a multiplicity of infection ( MOI ) of 3, resulting in infection of the majority of the cells, and harvested at 3, 5, and 8 hours post infection ( hpi ) . For each time point, we monitored transcriptome-wide translation levels by preparing ribosome profiling libraries from cells treated with the translation elongation inhibitor cycloheximide ( CHX ) . In parallel, we prepared RNA-seq libraries following poly-A selection to generate a detailed depiction of transcript levels. In addition, in order to measure total RNA levels and rRNA levels, we prepared RNA-seq libraries from total RNA without poly-A selection or rRNA depletion. Two independent biological replicates were prepared for all libraries from uninfected cells and from cells infected with SARS-CoV-2 for 8 hours.To directly evaluate mRNA decay during SARS-CoV-2 infection we employed SLAM-seq on Calu-3 cells infected with SARS-CoV-2 as well as uninfected cells. This approach allows to measure endogenous mRNA half-lives based on 4-thiouridine ( 4sU ) incorporation into new RNA during transcription. After RNA extraction, 4sU is converted to a cytosine analogue using iodoacetamide, and these U to C conversions are identified and quantified by RNA sequencing. The cells were infected with SARS-CoV-2 BavPat1/2020 Ref-SKU: 026V-03883 at a multiplicity of infection ( MOI ) of 3, resulting in infection of the majority of the cells, or left uninfected, labeled with 4sU at 3hpi and harvested at 4, 5, 6 and 7 hpi. Following iodoacetamide-conversion RNA was used to prepare poly-A selected libraries. Two biological replicates were prepared from uninfected cells and infected cells at 5hpi, following 2 hours of 4sU labeling. To test if SARS-CoV-2 interferes with nuclear mRNA export, Calu3 cells were left uninfected or infected with SARS-CoV-2 and subcellular localization of polyadenylated transcripts was assessed by cytoplasmic/nuclear (Cyto/Nuc) fractionation followed by RNA-seq. RNA was extracted from nuclear and cytoplasmic fractions in parallel to whole cell from the same samples. Two biological replicates were prepared from all samples.