Fig. 9: DDX3X promotes optimal pUs3 virion incorporation.

a HeLa cells were grown on 245 mm plates and treated with control non-targeting NC1 or siDDX3X for 48 h. Transfected cells were mock-treated or infected with wild-type strain 17⁺ virus. At 24 hpi, supernatant containing the mature virions were collected and concentrated at 60,000 x g. They were next analyzed on 5–20% gradient SDS-PAGE and Western blotting, using a panoply of antibodies targeting various viral tegument proteins. Total lysates of the same cells collected in lysis buffer and analyzed in parallel served as negative (mock cells) and positive (infected cells) antibody controls. DDX3X depletion in the cell and virions was confirmed by probing DDX3X in the same samples. b Image Lab version 5.0 was then used to quantify the Western blots (note that detection of the proteins was done on an instrument with a 4-log linear range, not on X-ray film). VP5, which served as a loading control, was used to normalize the data of the infected samples. The bars indicate means of three independent experiments and the standard error of the means (bilateral Student T-tests; *p < 0.05). pU_S3 was the only statistically different tegument in the DDX3X depleted virions.