Nuclear targeting of adenovirus type 2 requires CRM1-mediated nuclear export

Abstract

Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.

Figure 1.

LMB and RJA inhibit adenovirus-mediated gene expression. TC7 (A and C) or HeLa (B and D) cells were treated with LMB (A and B) or RJA (C and D), incubated with Ad5-luc in the cold for 60 min (2.9 × 10⁴ particles per cell), washed, and analyzed for luciferase activity 4 h postinfection. The mean values of triplicate samples from one typical experiment are shown as relative light units (rlu), normalized to the number of cells, including the SEs of the mean from triplicate samples. (E) The average fold inhibition of normalized luciferase activity in TC7, HeLa, A549, 911, KB, COSN, CV1, A431, normal human foreskin fibroblasts (2N), and HUVEC cells treated with 20 nM LMB, compared with non–drug-treated cells. The mean values of triplicate samples are shown. Independently, the subcellular localization of Ad2-TR in the 20 nM LMB-treated cells was scored: (+) indicating MTOC-arrest, (-) random cytoplasmic distribution and not determined phenotypes (n.d.). The viability of the cells treated with 20 nM LMB and infected with Ad5-luc was measured by metabolic labeling with [³⁵S]methionine (F).

Figure 2.

Knockdown of cellular CRM1 by siRNA reduces nuclear targeting of Ad2. (A) HeLa cells were transfected with siRNA specific for CRM1 or nonsilencing siRNA. Cell lysates were analyzed by Western blotting using anti-CRM1 (a) and anti-calnexin antibodies (b), respectively. (B) CRM1 specific siRNA (red) or nonsilencing siRNA (black) transfected HeLa cells were infected with eGFP-expressing Ad at 46 h posttransfection, immunolabeled for CRM1 detection, and analyzed by flow cytometry of eGFP fluorescence (a) and CRM1 (b). (C) CRM1-siRNA and nonsilencing siRNA-transfected cells were incubated with Ad2-TR in the cold for 1 h, warmed for 40 min, pulsed with transferrin-647, fixed 80 min postinfection, and immunolabeled for CRM1. The nuclei are stained with DAPI and DIC images are included for reference. Projections of entire stack of CLSM images are shown. Bar, 10 μm.

Figure 3.

LMB and RJA inhibit the nuclear export of 2xNES-eGFP and nuclear targeting of adenovirus. TC7 cells transfected with plasmid DNA encoding 2xNES-eGFP for 24 h were treated or not treated with 20 nM LMB or 10 ng·ml^-1 RJA, incubated with Ad2-TR in the cold, and warmed for 0 or 80 min. Entire stacks of CLSM images are shown for 2xNES-eGFP (NES-GFP, green) and Ad2-TR (red), and sections across the cell center for DAPI stainings of DNA. Bar, 10 μm.

Figure 4.

Adenovirus arrests at the MTOC of LMB-treated TC7 cells. (A) Cells were pretreated with or without 20 nM LMB in growth medium for 30 min, exposed to Ad2-TR in the cold for 60 min, warmed for indicated times with or without LMB, and fixed with pFA (at 0 min postinfection pi) or methanol (40 and 80 min postinfection). The MTOC was immunolabeled by with an γ-tubulin antibody. The projections of the complete stacks of confocal sections are shown. DIC images are included as a reference. Bar, 10 μm. (B) Thin-section electron micrographs across the centrosomes of Ad2-infected LMB-treated TC7 cells 120 min postinfection. Note the localization of viral particles (arrowheads) in the pericentriolar matrix near the centrioles (arrows).

Figure 5.

The MTOC arrest of adenovirus in LMB-treated TC7 cells requires MTs and also occurs in mitotic cells. (A) TC7 cells were treated with or without LMB (20 nM) and nocodazole (noc, 20 μM), infected with Ad2-TR for 80 min, fixed with PHEMO fix, and immunostained for α-tubulin (green) and DAPI (blue). DIC images are included as a reference. Bar, 10 μm. (B) Synchronized mitotic cells treated with or without LMB were infected with Ad2-TR (red) for 60 min, fixed with PHEMO fix, and stained for α-tubulin (green) and DNA (DAPI, blue). Whole-cell CLSM analyses are shown. Note the enrichment of Ad2-TR at spindle pole bodies of LMB-treated mitotic cells. Bar, 10 μm.

Figure 6.

Inhibition of nuclear envelope targeting of adenovirus in TC7, A549, and HeLa cells. Quantitative subcellular analyses of Ad2 by thin-section EM in LMB-treated and control TC7, A549, and HeLa cells. Viral particles (v.p.) were counted at the nuclear membrane (A), in the cytosol (B), endosomes (C), and at the plasma membrane (D). Results indicate the means and SE from the number of analyzed cells and v.p.

Figure 7.

LMB inhibits adenovirus capsid disassembly and nuclear import of protein VII and viral DNA. LMB-treated and control TC7 (A) and HeLa (B) cells were infected with Ad2 for 150 min, fixed, and immunostained for the disassembled capsid protein hexon and the DNA-associated core protein VII. The merged stacks of the entire set of CLSM sections are shown. Bar, 10 μm. (C) LMB blocks Ad2 DNA import into the nucleus. TC7 cells were infected with Ad2 in the presence or absence of LMB for 180 min, processed for in situ hybridization, and immunostained for lamins A/B/C. Single CLSM sections across a central plane of the cells are shown including DIC. Bar, 10 μm.