Decrease of NAD RNA capping leads to increased HIV-1 infectivity.
(A) Cleavage positions of the NAD RNA cap by the three known decapping
enzymes Nudt12, Nudt16 and DXO. (B) In vitro DXO cleavage of NAD-U1
and TMG-U1 at various time points time (0, 10, 30, 60, 120 and 240
min)). (C) Quantification of DXO decapping activity based on PAGE
gels (experiments performed in triplicate, error bar represents the
standard deviation of the mean). (D) Western blot analysis of DXO
and GAPDH (as a control) in control cells and cells with overexpressed
(DXO OE). (E) HIV-1 infectivity determined in control cells and cells
with overexpressed DXO (DXO OE) (ANOVA test). (F) LC–MS quantification
of the NAD RNA cap level in sRNA isolated from control cells and cells
with overexpressed DXO (DXO OE) (t-test). (G) LC–MS
quantification of the NAD RNA cap level in pulled-down U1 RNA (t-test). All the experiments were performed at least in
biological duplicates.