Fig. 3: The DDX3X carboxyl tail DDX3X physically interacts with the HSV-1 nuclear egress protein pU_L31.

The interaction of DDX3X and pUL31 was assessed using co-IP. a HeLa cells were either mock-treated or infected with wild-type strain F virus (negative controls) or with HA-tagged pU_L31 virus at an MOI of 5 for 12 hpi. Cells were lysed and HA-tagged pU_L31 pulled down using anti-HA agarose beads. Total lysates and eluates were analyzed by WB by probing pU_L31 (anti HA or pU_L31) and DDX3X (anti-human DDX3 rabbit R648 polyclonal serum) as indicated to the left of the blots. b The reverse co-IP was also performed with HeLa cells either mock-treated or infected as above but by precipitating DDX3X (anti-human DDX3 rabbit R648 polyclonal serum and protein A agarose beads). The presence of DDX3X and pU_L31 in the eluates were assessed by Western blot using anti-DDX3X R648 or anti-pU_L31 as shown to the left of those blots. c Schematic map of DDX3X showing its different domains and motifs (adapted from¹⁰³). IDR, NTE and CTE represent intrinsically disordered domains, N- and C-terminal extensions respectively. The helicase core (residues 182-544) represents the minimally active portion of DDX3X. All conserved DDX3X motifs are colour coded as defined by the legend on the figure. These motifs modulate ATP and RNA binding or the coordination between the two functions. d Recombinant GST-tagged DDX3X constructs or a GST alone control were captured on glutathione Sepharose 4B beads. HeLa infected lysates (HA-tagged pU_L31 virus, MOI of 5, 24 hpi) were pre-cleared on the GST preloaded glutathione Sepharose beads then incubated with the various enriched GST-DDX3X constructs for 3 h. The beads were boiled in sample buffer to release the bait. Samples were analyzed on 5–20% gradient SDS-PAGE containing 0.5% TCE. The level of the bead bound GST-tagged constructs was analyzed by UV exposure (TCE panel) on a ChemiDoc (BioRad). Binding of pU_L31 was evaluated by Western blotting using a HA-tag antibody (bottom panel). Stars highlight the position of each GST construct. TL represents total lysates prior to its loading on the beads. e Quantification of pU_L31 binding to the beads for each DDX3X construct (drawn schematically). Binding was calculated by normalizing the HA-tagged pUL31 Western blot signal to the TCE signal for each GST chimera. Fold changes were measured by arbitrarily setting the binding to GST-DDX3X aa 623-662 to one. Absence of binding is defined as NB (no binding). ±Standard error of the means (bilateral Student T-tests; *p < 0.05). The figure represents four independent experiments.