The SARS-CoV-2 nucleocapsid protein interferes with the full enzymatic activation of UPF1 and its interaction with UPF2

Figure 1.. SARS-CoV-2 Np directly interacts with UPF1 in cellulo and in vitro. (A) Co-immunuprecipitation of endogenous UPF1 with SARS-CoV-2 Np in HEK 293T cells. Western blot of UPF1 and strep-tagged SARS-CoV-2 Np before (input, left panel) and after immunoprecipitation (right panel) of endogenous UPF1. As a negative control, immunoprecipitation was also performed using a control serum. (B) Schematic diagram showing the SARS-CoV-2 Np, UPF1 and UPF2 domains used for this study. Structural domains are represented by rectangles and (predicted/confirmed) flexible regions by black lines. (C) Pulldown experiment using CBP-UPF1-CH-HD (lane 1), CBP-UPF1-HD (lane 2) or CBP-eIF4A3 (lane 3) as bait. After incubation, protein mixtures before (input 20% of total) or after precipitation (precipitate) were separated on a 12% SDS-PAGE gel and visualized by Coomassie staining. (D) MST quantification of UPF1 (target) –Np (ligand) interaction. The graph depicts the titration of Np-L or UPF2(770–1204) to a constant amount of UPF1-HD or UPF1-CH-HD fluorescently labeled. The binding curve represents the ligand concentration in a logarithmic scale, versus the proportion of the target that is bound by the ligand…

Figure 2.. SARS-CoV-2 Np inhibits UPF1 unwinding activity and cellular NMD. (A) A representative gel showing ATP-induced unwinding of the dsDNA substrate with 5′ overhangs of 12 nucleotides by UPF1-HD, the UPF1-HD:Np-L, UPF1-HD:Np-NTD and UPF1-HD:Np-CTD preformed complex, and Np-L, Np-NTD and Np-CTD alone. The substrate and the experimental workflow are depicted at the top of the gel. In the control lanes, ATP was replaced by ADPNP or samples were heat-denatured (95°C). (B) Graph showing the fraction of DNA oligonucleotide released over time. Data points derived from four independent experiments were fitted to the pseudo-first order equation y = A[1-e(-kt)], where A and k represent, respectively, the amplitude and the rate constant of the unwinding reaction. (C) NMD assay was performed in cells overexpressing or not the SARS-CoV-2 Np. In the upper panel, the western blot displays the overexpression levels of SARS-CoV-2 Np. NMD efficiency was assessed by the quantitative detection of NMD reporter mRNAs at steady state. Relative levels of Gl-WT and Gl-PTC mRNAs were determined by RT-qPCR. The statistical significance was supported by a T test with three independent biological replicates. (D)…

Figure 3.. SARS-CoV-2 Np does not affect the ATPase and protein-displacement functions of UPF1. (A) Graph showing the percentage of ATP hydrolyzed as a function of time by the UPF1–HD, UPF1–HD:Np-L-preformed complex, and Np-L alone under conditions of steady-state ATPase turnover (see ‘Materials and methods’ section). (B) Time course of an active displacement of streptavidin from a 3′ biotinylated DNA by UPF1-HD, the UPF1-HD:Np-L preformed complex, and Np-L alone. The pipeline of the experiment is showed at the top of the gel. At various times (see Methods section) aliquots from the helicase reaction were mixed in a quench buffer and loaded immediately on 8% polyacrylamide gel containing 0.3% of SDS to selectively denature protein/s:DNA complexes without altering biotin–streptavidin interaction. On the right, graph showing the fraction of streptavidin displaced as a function of time (min). The bound product fraction at time 0 is calculated using Eq. F0 = P0/(R0 + P0) and the product fractions at time ‘t’ using Eq. Ft=(Pt-F0(Pt + Rt))/((1-F0)(Pt + Rt)) where P is the intensity of the band corresponding to the reaction product (biotinylated DNA) and R the intensity of the substrate…

Figure 4.. SARS-CoV-2 Np interacts with UPF1 and UPF2 affecting their interaction. (A) Pulldown experiment using CBP-UPF1-CH-HD (lanes 1 to 4) as bait and UPF2(770–1204), SARS-CoV-2 Np and eIF4A3 as preys. After incubation, protein mixtures before (input 20% of total) or after precipitation (precipitate) were separated on a 12% SDS-PAGE gel and visualized by coomassie staining. (B) Pulldown experiment using CBP-UPF1-CH-HD (lanes 1–8) as bait and UPF2(770–1204) (lane 1) and SARS-CoV-2 Np (lane 5) as preys. Three increasing amount of Np (lanes 2 to 4) or UPF2(770–1204) (lanes 6 to 8) were added to fixed amount of UPF1-UPF2 or UPF1-Np. (C) MST quantification of UPF2 (target) – Np or -UPF1 (ligands) interactions. The graph depicts the titration of Np-L or UPF1-CH-HD to a constant amount of UPF2(770–1204) fluorescently labeled. The binding curve represents the ligand concentration in a logarithmic scale, versus the proportion of the target that is bound by the ligand (fraction bound). The binding curve was fitted using a non-linear regression model to calculate the dissociation constant (KD). Red and blue curves are UPF2(770–1204) versus Np-L or UPF1-CH-HD respectively. Data points represent the…

Figure 5.. UPF1 and UPF2 have antagonistic effects on SARS-CoV-2 replication. (A) Schematic representation of the experiments performed with Vero E6 cells infected with a replicative strain of SARS-CoV-2. (B) An NMD assay was conducted on mock-infected or SARS-CoV-2-infected cells, as indicated by NeonGreen fluorescence expression (upper panel). Cl-WT and Gl-PTC mRNAs were quantified by qRT-PCR, and their relative levels were plotted in a graph. Statistical significance was determined using a T-test based on three independent biological replicates. (C) Vero E6 were transfected with DNA coding for HA-UPF1, HA-UPF2 or a control DNA. Twenty-eight hours later they were infected at a MOI of 0.1 with a Neon-Green bearing replicative-competent SARS-CoV-2 virus. The extent of viral replication and the overexpression of UPF1/ UPF2 were assessed 18 hours post-infection using flow cytometry. The graph presents averages and SEM from four independent experiments. * P value < 0.05, following an ordinary one-way Anova test between the indicated conditions. (D) Proposed mechanism of action. Upon entering the cell, the SARS CoV-2 genome is initially covered by Np shielding its genetic material from…