FIG 5.

SARS-CoV-2 hijacks DDX6 for viral replication. (A) Dynamic redistribution of DDX6 in response to SARS-CoV-2 infection. Vero E6 TMPRSS2 cells at the indicated times after inoculation of SARS-CoV-2 were stained with anti-SARS-CoV-2 nucleocapsid (ab273434 [6H3]) and anti-DDX6 (A300-460A) antibodies. (B) SARS-CoV-2 does not induce stress granule formation. Uninfected Vero E6 TMPRSS2 or SARS-CoV-2-infected cells at 24 h postinfection were incubated at 37°C. Uninfected cells were also incubated at 43°C for 45 min. Cells were then stained with anti-SARS-CoV-2 nucleocapsid and anti-G3BP1 (A302-033A) antibodies. (C) Host protein expression levels in response to SARS-CoV-2 infection. The results of the Western blot analysis of cellular lysates with anti-SARS-CoV-2 spike (GTX632604 [1A9]), anti-DDX6, anti-G3BP1, or anti-β-actin antibody in the SARS-CoV-2-infected Vero E6 TMPRSS2 or the HEK293T ACE2 cells at 24 h postinfection at an MOI of 0.5 as well as in the uninfected cells are shown. (D) Requirement of DDX6 for SARS-CoV-2 infection. Inhibition of endogenous DDX6 protein expression by the shRNA-producing lentiviral vector. The results of Western blot analysis of cellular lysates with anti-DDX6 or anti-β-actin antibody are shown. The level of intracellular SARS-CoV-2 RNA in the cells at 72 h postinfection at an MOI of 0.5 was monitored by real-time LightCycler RT-PCR. Results from three independent experiments are shown. The level of SARS-CoV-2 RNA in the DDX6 knockdown cells was calculated relative to the level in HEK293T ACE2 cells transduced with a control lentiviral vector (Con). *, P < 0.05 compared to control cells.