Immunofluorescence They seeded HeLa cells, HeLa-FRT UBAP2L KO cells, or parental cells in six-well dishes with coverslips at 25% confluency. The next day, they transfected the cells with 250 ng of DNA and 1 μL of jetOPTIMUS overnight in DMEM media supplemented with FBS (10%) and PenStrep (1%). They then changed the media to media containing 0.5 μM sodium arsenite for 30 minutes to induce stress granule formation. After washing with PBS, they fixed the cells for 20 minutes with 4% formaldehyde in PBS. They permeabilized the cells for 10 minutes with PBS containing 0.5% Triton-100. Following three 5-minute washes with PBS-T (0.05% Tween), they incubated the cells with 25 mM Glycine overnight at 4 °C. They blocked the coverslips in TBST (0.05% Tween) containing 3% BSA for 45 minutes at room temperature. They incubated primary antibodies at a 1:400 dilution in 3% BSA TBST (0.05% Tween) overnight at 4 °C. After three 5-minute washes with TBST (0.05% Tween), they incubated the coverslips with secondary antibodies for 1 hour at room temperature. They mounted the coverslips in MOWIOL mounting solution and imaged them on a Delta-Vision Elite microscope with a 60x oil objective. They analyzed the data in Fiji and plotted it with a Prism 9 GraphPad software. Live-Cell Imaging They seeded HeLa cells at 25% confluency in six-well dishes and transfected them the following day with 200 ng of DNA and 1 μL of jetOPTIMUS overnight in 3 mL media. They changed the media the next day and seeded the cells in eight-well ibidi dishes at 40% confluency. They took pictures every 5-10 minutes in 2 z-stacks on a Delta-Vision Elite microscope with a 60x oil objective. They filmed the cells 48 hours post-transfection for 10 minutes and added sodium arsenite to a final concentration of 0.5 μM. They filmed the cells for additional hours to follow the formation of stress granules. They analyzed the data in Fiji and plotted it with a Prism 9 GraphPad software. Immunoprecipitation They seeded cells at 25% confluency in 15-cm3 dishes with 2 μg of DNA and 2 μL of jetOPTIMUS overnight in 15 mL media. They changed the media the following day and collected the cells after 48 or 24 hours post-transfection. They lyzed the cell pellets from each 15-cm3 dish in 350 μL lysis buffer (100 mM NaCl, 50 mM Tris pH 7.4, 0.1% NP40, 0.2% Triton-100, 1 mM DTT) supplemented with protease and phosphatase inhibitor tablets. They sonicated the lysate at 4 °C for ten cycles of 30 seconds ON, 30 seconds OFF using a Bioruptor sonicator. Following sonication, they cleared the lysates for 45 minutes at 20,000 × g. They collected the supernatants and measured their concentrations. They incubated the lysates with 10 μL pre-equilibrated GFP-trap beads for 1 hour at 4 °C on a rotor wheel. They washed the beads three times with 800 μL wash buffer (150 mM NaCl, 50 mM Tris pH 7.4, 0.05% NP40, 5% glycerol, 1 mM DTT). If the immunoprecipitations were prepared for mass spectrometry analysis, they performed one additional wash with basic wash buffer (100 mM NaCl, 50 mM Tris pH 7.4, 5% glycerol). If the immunoprecipitations were analyzed by SDS-PAGE and western blot, they used 25 μL 2× LSB to elute the samples. They boiled the samples and separated them by SDS-PAGE and processed them for western blot using the following antibodies: FMR1 rabbit, FXR1 mouse, c-myc, GFP rabbit, G3BP1, and UBAP2L rabbit.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.