Extended Data Fig. 5: Characterization of HIV-1 RNA transfected in U937 cells.
From: FTSJ3 is an RNA 2′-O-methyltransferase recruited by HIV to avoid innate immune sensing
a, HIV viruses were produced in 293T cells transfected with FTSJ3 siRNA or a control non-specific siRNA (scramble). Inhibition of FTSJ3 was assessed by western blot. The experiment was repeated more than 10 times with similar results or a more efficient knockdown. b, Purified recombinant wild-type or catalytic KDKE mutant FTSJ3–His was incubated with in vitro-transcribed HIV-1 RNA in the presence of radiolabelled [3H]-SAM, and MTase activity was determined by [3H]-methyl incorporation. n = 3 independent samples; results shown as the mean ± s.d. representative of two independent experiments. Indicated P values obtained by two-tailed Student’s t-test. c, Purified recombinant WT-FTSJ3 was incubated without exogenous RNA or with the 8362–8461 HIV RNA fragment and MTase activity was determined by [3H]-methyl incorporation assay (left) or 2D TLC separation (right). [3H]-methyl incorporation assay results were normalized by subtracting the background obtained in the absence of exogenous RNA. 2D TLC separation of 5′-[32P]-NMPs resulted from nuclease P1 hydrolysis of HIV RNA transcript 8362–8461 radiolabelled with [α-32P]ATP and incubated with WT-FTSJ3 or mutant KDKE-FTSJ3 recombinant proteins. Migration was performed in the N1/N2 or N1/R2 combination of solvents (see Methods). Positions of unmodified AMP (pA), 2′-O-methyl AMP (pAm) and inorganic phosphate (Pi) are indicated. The experiment was repeated twice with similar results.
