Global mapping of the SARS-CoV-2 microRNA interactome

Using the CLEAR-CLIP methodology (PMID: 26602609) combined with high throughput sequencing, all the binding sites of the Argonaute (AGO) proteins on SARS-CoV-2 RNAs were identified as well as the miRNAs contributing to each binding site. Consequently, SARS-CoV-2 mutants were engineered to investigate the functional relevance of the main binding sites. The effect of the infection on the cellular miRNAs was also investigated using the same CLEAR-CLIP data and RNA-Seq analysis. The work was done with the SARS-COV-2 virus strain DK-AHH1 (PMID: 33903110). Monkey kidney VeroE6 cells and human lung A549hACE2 cells were used as model systems for infection. All experiments were performed with a multiplicity of infection (MOI) of 0.001 and the cells collected for experiments 48 hours after the initiation of infection. The experimental setup for CLEAR-CLIP was as follow for each cell line: 1.5-2 million cells (VeroE6) or 2.5 million cells (A549hACE2) were seeded in each of 8 x 10 cm dishes. The day after, the cells in 5 x dishes were infected. Two days later, the cells of all 8 x dishes were collected and processed for CLEAR-CLIP independently for each dish but in parallel. Among the 5 x infected samples, one was processed without cross-linking, another was processed using a non-specific antibody instead of the pan-AGO antibody. This resulted in 3 x infected replicates (NFO147_A-C for VeroE6; NFO175_A-C for A549hACE2), 3 x non-infected replicates (NFO147_F-H for VeroE6; NFO175_F-H for A549hACE2), 1 x non-cross-linked infected control (NFO147_D for VeroE6; NFO175_D for A549hACE2) and 1 x non-specific-immunoprecipitation infected control (NFO147_E for VeroE6; NFO175_E for A549hACE2). Loss-of-binding (1L: C10039G + A10042U; 2L: U11377A + U11380A; 3L: C17462G + U17466C; 4L: U19869A + G19872C; 5L: U25434A + G25437A; 6L: U26732A + C26735U) ang Gain-of-binding (3G: A17465U; 5G: U25435G) SARS-CoV-2 mutants were engineered for six mains miRNA binding sites identified and CLEAR-CLIP performed for each of them in both cell lines (NFO181 samples). Furthermore, an RNA-Seq experiment was performed for 3 x replicates infected with wild-type SARS-CoV-2 and 3 x non-infected replicates for each cell lines (NFO190). For this last experiment, samples were also collected 72 hours after the initiation of infection for the A549hACE2 cells.