Cap-independent translation and a precisely located RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response

Figure 1.. Expression of NSP1 destabilizes host mRNAs. (A) HEK293 cells were transfected with plasmids encoding either eGFP or HA-NSP1 (4 μg DNA per 10-cm dish) and collected after 24 h. Isolated RNA was subjected to poly(A)-dependent enrichment, quantified, and plotted in a relative manner; n = 5, the bar represents SE. (B) Cells transfected as detailed in (A) were treated with Actinomycin D (7.5μg/ml) for 0, 2, 4 and 6 h, harvested and subjected to MARS-seq procedure to determine half-lives of polyadenylated RNAs; n = 2. (C) Changes in half-lives of multi- and single-exon mRNAs upon expression of HA-NSP1; n = 2. (D) HEK293 cells were transfected with plasmids encoding Renilla luciferase (Rluc) reporter genes either with or without intron in the 5′UTR (upper panel) along with plasmids encoding for eGFP or HA-NSP1. After 24 h, the relative expression of both reporters was calculated and plotted; n = 3, bars represent SE.

Figure 2.. The impact of NSP1 on translation and survival. (A) HEK293 cells were transfected with plasmids encoding HA-NSP1 or eGFP, as a control. After 24 h, the cells were collected, lysed and subjected to polysomal profiling. The left panel displays continuous UV absorbance of both gradients of one out of two independent experiments. The middle panel shows the signal distribution between the different grouped fractions. The right panel directly compares the polysomal vs. monosomal fractions. In all panels, bars are SD of n = 2. See also Figure S2A for analysis of MCF7 cells. (B) HEK293 cells were transfected with plasmids encoding either eGFP or HA-NSP1. After 24 h, puromycin (10 μg/ml) was added for 5 min, and the cells were collected on ice, lysed and 50 μg of total lysates were resolved on 10% SDS-PAGE and probed with anti-puromycin antibodies. The relative signals obtained from the different lanes were analyzed and plotted on the right panel; n = 4, bars represent SD. See also Figure S2B for the original images and S2C for expression of specific proteins. (C) MCF7 cells were transfected with plasmids encoding either eGFP or HA-NSP1 and after 24 h lysed and subjected to polysomal…

Figure 3.. Identification of RNA sequence that protects from NSP1-mediated repression. (A) MRC5 cells were transfected with plasmids encoding for eGFP, control 5′UTR-HA-NSP1 or CoV2-5′UTR-HA-NSP1 with its native 5′UTR immediately following the TSS (which is termed HA-NSP1 in this study). After 24 h, the cells were collected and 50 μg from the extracted total proteins were resolved on 9% SDS-PAGE and probed to detect the indicated proteins. (B) MRC5 cells were transfected with plasmids encoding the control 5′UTR-HA-NSP1 or CoV2-5′UTR-HA-NSP1, as detailed in (A), harvested after 24 h and split into two equal parts. One part was subjected to RNA isolation and subsequent RT-qPCR analysis to determine mRNA levels. The second part of each harvest was dedicated to the resolution of total proteins (50 μg) on SDS-PAGE and detection of the indicated proteins. Relative values of each biological repeat (n = 3) were plotted, the bars represent SE. (C) Impact of NSP1 on the expression of reporter mRNAs. MRC5 cells were transfected with plasmids encoding either eGFP or TSS-5′UTR-HA-NSP1. After 20 h, the indicated mRNA reporters bearing both 5′cap and poly(A) tail were transfected, and the luciferase…

Figure 4.. The translational features of SARS-CoV-2 5′UTRs. (A) Upper panel: schematic representation of the bi-cistronic assay. MCF7 (left panel) or MRC5 (right panel) cells were transfected with bi-cistronic plasmids encoding for the SARS-CoV-2 genomic 5′UTR or EMCV-derived IRES element as a positive control. Both elements were introduced in their correct or flipped (i.e. anti-sense) configuration as a control for non-specific activity. Cells were grown for 24 h in standard medium (NT) or supplied with Torin1 (25 nM), after which Rluc and Ffly activities were assayed; n = 4, bars represent SE. (B) MRC5 cells were transfected with the indicated uncapped (PPP) in-vitro transcribed mRNAs bearing poly(A) tails. Rluc activity was tested 7 h later and is presented relative to the control plasmid-derived 5′UTR; n = 4, the bars represent SE. (C) MCF7 and MRC5 cells were transfected with i) the indicated bi-cistronic plasmids and ii) plasmid encoding either HA-NSP1 or eGFP. Luminescence was assayed 24 h later, n = 4, the bars represent SD. (D) MRC5 cells were transfected with plasmids encoding either HA-NSP1 or eGFP. After 24 h, uncapped RNAs with poly(A) tails encoding for indicated 5′UTRs and a…