MOV10 Helicase Interacts with Coronavirus Nucleocapsid Protein and Has Antiviral Activity

FIG 1. Colocalization of MOV10 and N protein in MERS-CoV-infected cells. Confocal immunomicroscopy analysis was performed on Huh-7 cells that were mock infected or infected with MERS-CoV at an MOI of 0.1. At 20 hpi, cells were fixed and stained with antibodies specific for MOV10 (green; left) and either (A) MERS-CoV N protein (red; middle) or (B) CoV nsp8 protein (red; middle). Cell nuclei, stained with Hoechst 33342 (blue; right), are shown in merged layers. (C) Pearson’s correlation coefficients. The data represent the medians from 20 cells in two independent experiments. Error bars represent SDs. ***, P < 0.001.

FIG 2. MERS-CoV N protein interacts with MOV10 and related cellular partners. Huh-7 cells were mock infected or infected with MERS-CoV at an MOI of 0.1 (Inf.) for 20 h. (A) Cell lysates were immunoprecipitated with anti-MOV10 antibody. (B) Cell lysates were immunoprecipitated with anti-MOV10 antibody with or without RNase treatment. (C) Schematic diagram of the interaction between MOV10 and selected cellular partners (yellow) involved in different RNA metabolism pathways (light blue). (D) Cell lysates were immunoprecipitated with anti-TIAR, anti-AGO2, or anti-UPF1 antibodies. (E) Cell lysates were immunoprecipitated with anti-ATP5B antibody. In all cases, protein presence in the original extract (input) or in the immunoprecipitated samples (IP) was detected by Western blotting using specific antibodies.

FIG 3. MOV10 activity on MERS-CoV infected cells. (A) Huh-7 cells were transfected with negative-control siRNA (siNeg; black) or MOV10-specific siRNAs (siMOV10; green), and the accumulation of MOV10 mRNA was quantified by RT-qPCR at the indicated time points. (B) Viral gRNA (blue) and sgmRNA-N (red) accumulation, at the indicated time points, was quantified by RT-qPCR in cells transfected with siMOV10 or with siNeg. (C) Cytoplasmic extracts were obtained from cells transfected with siNEG or siMOV10 and mock infected (0) or infected with MERS-CoV at an MOI of 1 for 8, 18, and 24 hpi. MOV10 and MERS-CoV N protein were detected by Western blotting. GAPDH was used as a loading control. Numbers under the blots indicate the estimated levels of N protein, normalized to GAPDH levels and relative to nonsilenced cells at 18 hpi. (D) Virus titers obtained from Huh-7 cells transfected with siNeg (gray) or siMOV10 (green) and infected with MERS-CoV. The values are means from three independent infections; error bars represent SD. ***, P < 0.001.

FIG 4. MOV10 activity on MERS-CoV infected cells overexpressing MOV10. (A) Huh-7 cells were transfected with an empty vector (pcDNA) or with a plasmid expressing MOV10 (pcDNA-MOV10) and infected with MERS-CoV at an MOI of 1. Viral gRNA (blue) and sgmRNA-N (red) were quantified by RT-qPCR at 24 hpi. (B) Cytoplasmic extracts were obtained from cells transfected with an empty vector (pcDNA) or with a plasmid expressing MOV10 (pcDNA-MOV10) and infected with MERS-CoV at a MOI of 1 for 18 and 24 hpi. MOV10 and MERS-CoV N protein were detected by Western blotting. GAPDH was used as a loading control. Numbers under the blots indicate the estimated levels of N protein, normalized to GAPDH levels and relative to pcDNA transfected cells at 18 hpi. (C) Virus titers obtained from Huh-7 cells transfected with an empty vector (pcDNA; gray) or with a plasmid expressing MOV10 (pcDNA-MOV10; green) and infected with MERS-CoV. The values are means from three independent infections; error bars represent SD. *, P < 0.05; **, P < 0.01.

FIG 5. RNA-IP of MOV10 complexes. Huh-7 cells were either mock infected or infected (Inf.) with MERS-CoV at a MOI of 0.1. At 20 hpi, RNA-protein complexes were immunoprecipitated with anti-MOV10 or negative-control anti-GFP antibodies. RNAs eluted from immunoprecipitated RNA-protein complexes were analyzed by RT-qPCR to quantify the associated viral gRNA (blue) and mRNA N (red). The values are means from three independent infections; error bars represent SD.

FIG 6. Effect of MOV10 absence on N protein interaction with related cellular partners. Huh-7 and MOV10-KO cells were mock infected or infected with MERS-CoV at a MOI of 1. (A) At the indicated time points, viral gRNA (blue), mRNA N (red), and MOV10 mRNA (gray) accumulation was analyzed by RT-qPCR. (B) In addition, cytoplasmic extracts were analyzed by Western blotting for detection of MOV10 and N protein. GAPDH was used as a loading control. Numbers under the blots indicate the estimated levels of N protein, normalized to GAPDH levels and relative to Huh-7 cells at 18 hpi. (C) Virus titers obtained in Huh-7 (blue) and MOV10-KO (red) cells infected with MERS-CoV. The values are means from three independent infections; error bars represent SD. *, P < 0.05. (D) MOV10-KO cells were mock infected or infected with MERS-CoV (Inf.) at an MOI of 0.1. At 20 hpi, cell lysates were obtained and immunoprecipitated with anti-TIAR, anti-AGO2, or anti-UPF1 antibodies. Proteins in the original extract (input) or in the immunoprecipitated samples (IP) were detected by Western blotting using specific antibodies.

FIG 7. MOV10 helicase activity is required for its antiviral function. (A) Schematic representation of wild-type (WT) and helicase mutant (HEL*) MOV10 proteins. CH, CH-rich domain; HEL, helicase domain; ATP bd, ATP binding motif; DEAG, conserved DEAG box; Ab, domain recognized by MOV10 antibody. The asterisks indicate the point mutations introduced. (B) MOV10-KO cells were transfected with an empty plasmid or plasmids expressing either WT or HEL* proteins and infected with MERS-CoV at an MOI of 1 for 18 and 24 hpi. MOV10 and N protein accumulation in cytoplasmic extracts was detected by Western blotting. GAPDH was used as a loading control. Numbers under the blots indicate the estimated levels of N protein, normalized to GAPDH levels and relative to pcDNA-transfected cells at 18 hpi. (C) Virus titers were measured at the indicated time points, from infected MOV10-KO cells previously transfected with an empty plasmid (gray) or plasmids expressing either WT (green) or HEL* (purple) proteins. The values are means from three independent infections; error bars represent SD. *, P < 0.05. (D) MOV10-KO cells were transfected with plasmids expressing either WT or HEL* proteins and then infected…

FIG 8. MOV10 interaction with N proteins from other human CoVs. (A) In all cases, cells were mock infected or infected with the corresponding CoV at an MOI of 0.1: DBT-ACE2 cells infected with SARS-CoV (left), Calu3 2B4 cells infected with SARS-CoV-2 (middle), and Huh-7 cells infected with HCoV-229E (right). At 20 hpi, cell lysates were obtained and immunoprecipitated with anti-MOV10 antibody. MOV10 and N proteins were then detected by Western blotting using specific antibodies. Huh-7 and MOV10-KO cells were infected with SARS-CoV-2, and both N protein accumulation (B) and viral titers (C) were analyzed at the indicated time points. Huh-7 and MOV10-KO cells were infected with HCoV-229E, and both N protein accumulation (D) and viral titers (E) were analyzed at the indicated time points. GAPDH was used as a cytoplasmic marker in all cases. Numbers under the blots indicate the estimated levels of N protein, normalized by GAPDH levels and relative to Huh-7 cells at 18 hpi. The values are means from three independent infections; error bars represent SD. ***, P < 0.001.