The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses (2022)
Figure 3

Figure 3. Characterization of DDX42 inhibitory activity against retroviruses and retroelements.

A. siRNA-transfected U87-MG/CD4/CXCR4 cells were infected with WT HIV-1 (NL4-3) and relative amounts of Minus Strand Strong Stop (MSSS), 1st and 2nd Strand Transfer DNAs, and nuclear forms of HIV-1 DNA (proviral DNA, and 2-LTR circles) were quantified by qPCR. DNAs from cells infected for 48 h in the presence of AZT and 3TC were used as a control. Mixed-effects analysis on log-transformed data with Dunnett's test.

B. Silencing efficiency in parallel samples from A.

C. Heat-annealed ODN/RNA 1-294 complex was incubated with HIV-1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analysed by PAGE 8% (P/T: primer/template; SSDNA: Strong-Stop DNA). A representative autoradiograph is shown.

D. Quantification of 3 biological replicates performed as in C. Two-way ANOVA on log-transformed data with Dunnett's test.

E. PLAs were performed in MDMs infected with HIV-1 or not (N.I. CTRL), using anti-Capsid and anti-DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale-bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD (n>65 cells per condition). Mann-Whitney test.

F. siRNA-transfected TZM-bl cells were infected with the indicated VSV-G-pseudotyped, replication competent viruses and β-galactosidase signals measured 24 h later. Two-way ANOVA on log-transformed data with Dunnett's test.

G. Silencing efficiency in parallel samples from F.

H. siRNA-transfected U87-MG/CD4/CXCR4 cells were infected with HIV-1- HIV-2- FIV- EIAV-based, GFP-coding LVs and infection efficiency was scored 24 h later by measuring the percentage of GFP expressing cells by flow cytometry. Two-way ANOVA on log-transformed data with Dunnett's test.

I. siRNA-transfected U87-MG/CD4/CXCR4 cells were infected with GFP-coding B-MLV retroviral vector and infection efficiency measured 24 h later by flow cytometry. Simple linear regression analysis.

J. HEK293T were co-transfected with GFP-coding LINE-1 plasmids (RPS-GFP or LRE3-GFP) or with an inactive LINE-1 plasmid (JM111) together with either a Firefly- or DDX42-coding plasmid. GFP expression was measured by flow cytometry 7 days later. Two-way ANOVA on log-transformed data with Sidak's test.

K. HEK293T were co-transfected with pRPS-GFP and a Flag-Firefly- (negative control) or Flag-DDX42-coding plasmid, followed by Flag immunoprecipitation and immunoblot analysis. A representative immunoblot is shown.

L. Left, RNA extraction and LINE-1 RT-qPCR on parallel samples from K. Two-way ANOVA on log-transformed data with Sidak's test. Right, Percentage of immunoprecipitated RNA. T-test on log-transformed data.

Data information: A-B and D-J, Data represent the mean ± S.E.M of 3 biological replicates, L, Data represent the mean ± S.E.M of 5 biological replicates. P values are denoted as follow: ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.