Phosphorylated LIC1 is required for transport of adenovirus to the nucleus. (A) Protein levels in lysates of A549 cells exposed to siRNAs (48 h) and expression of RNAi-resistant GFP-LIC1 (24 h) evaluated by immunoblotting using anti-LIC1 and anti-GFP antibodies, with anti-tubulin as a loading control. (B) LIC1 RNAi rescue of adenovirus redistribution. LIC1 siRNA-treated A549 cells transfected at 24 h with GFP-tagged LIC1 cDNAs (indicated on top) and infected at 48 h with Ad5 for 60 min. Cells were stained for Ad5, GFP, and DNA (DAPI). Transfected cells are outlined. LIC1 RNAi–inhibited redistribution is rescued by GFP-LIC1 wild type (wt) and GFP-T213D but not GFP-T213A. (Capsids outside the encircled cells are located in adjacent cells.) (C) LIC1 RNAi rescue effect on virus run length. A549 cells treated with LIC1 siRNA were subsequently transfected with LIC1 wild type, -T213D, or -T213A and infected with Alexa Fluor 546–labeled Ad5 48 h later and imaged for particle tracking (see Materials and methods). LIC1 wild type and phosphomimetic T213D mutant showed rescue in virus run length, in contrast to the LIC1 dephosphomutant T213A. Error bars show SDs. (D) LIC1 RNAi rescue of adenovirus redistribution like in B, in the presence of PKA inhibitor PKI-myr. The inhibitor alone (GFP) inhibited virus accumulation at the nucleus, whereas partial inhibition was observed in the RNAi rescue cells. Lines define cell outlines. (E) Quantification of virus redistribution shown in B and D. Mean percentage of Ad5 particles localizing at the nucleus 60 min p.i. ± SD from at least three independent experiments with >40 cells each. Blue, control condition; gray, summary of experiments for B; orange, summary of experiments for D. **, P < 0.01. ctr, control. Bars, 20 µm.