The leader protein of Theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins

Abstract

The leader protein of Theiler's virus was previously shown to block the production of alpha/beta interferon by infected cells. Here, we observed that expression of the leader protein in infected cells triggered subcellular redistribution of a nucleus-target green fluorescent protein. It enhanced redistribution of the nuclear polypyrimidine tract-binding protein but had no influence on the localization of the nuclear splicing factor SC-35. The leader protein also interfered with trafficking of the cytoplasmic interferon regulatory factor 3, a factor critical for transcriptional activation of alpha/beta interferon genes.

FIG. 1.

Perturbation of endogenous IRF-3 subcellular localization by the leader protein. Endogenous IRF-3 and viral antigen were detected by double immunofluorescence assay, in L929 cells infected for 9 h with KJ6 or TM659. The left panel shows endogenous IRF-3 staining in a cell infected with TM659, the virus expressing the mutant L^cys protein. Eleven percent of cells positive for viral antigen displayed the clear nuclear translocation of IRF-3 shown in the figure, whereas 89% of infected cells conserved the cytoplasmic staining seen in uninfected cells (data not shown). After infection with the KJ6 virus, expressing the wild-type L protein, 95% of cells positive for viral antigen showed IRF-3 redistribution through the cytoplasm and the nucleus (right panel). Note the conspicuous nuclear IRF-3 labeling with unstained nucleoles, visible in TM659-infected cells (left), and the heterogeneous nuclear IRF-3 labeling seen in KJ6-infected cells (right).

FIG. 2.

Redistribution of cytoplasmic and nuclear proteins by the leader protein in BALB/3T3 cells. Histograms show the percentages of infected cells showing nuclear, cytoplasmic, or both cytoplasmic and nuclear IRF-3 staining. (A) Endogenous IRF-3 detection in cells infected for 6 h with the indicated viruses. (B and C) Localization of the GFP-mIRF-3 fusion protein (B) and of the mutant protein lacking the NLS (C). Cells were counted 6 h after infection with DA1 (L^wt) or TM598 (L^cys). (D) Localization of the NLS-eGFP fusion protein in cells infected for 10 h with the indicated viruses. For each experiment, IRF-3 localization was observed by fluorescence microscopy in 100 cells positive for viral antigen. Histograms show the means and standard errors of the means for three independent experiments done in parallel for DA1 and TM598.

FIG. 3.

Redistribution of a cytoplasmic protein, IRF-3, lacking an NLS. BALB/3T3 cells stably expressing GFP-mIRF-3 (top row) or GFP-mIRF-3#NLS (third row) were left untreated and uninfected (leftmost panels, NI), treated for 30 h with 2.5 μg of poly(IC), infected for 8 h with NDV, or infected for 6 h with DA1 or TM598 as indicated. Immunofluorescence assays for viral capsid antigen were performed in the case of DA1 and TM598 infections. White arrows point to cells positive for viral antigen. Green fluorescence of GFP-mIRF-3 and GFP-mIRF-3#NLS fusions is shown. Note that, upon poly(IC) transfection or NDV infection, only the IRF-3 fusion containing the NLS was translocated to the nucleus, whereas upon infection with DA1 both fusion proteins were redistributed.

FIG. 4.

Redistribution of NLS-eGFP but not of SC-35. (A) BALB/3T3 cells expressing NLS-eGFP were infected with the DA1 and TM598 viruses. Ten hours after infection, cells were fixed and stained for viral antigen (VP1). White arrows point to cells positive for viral antigen. NLS-eGFP green fluorescence (left panels) was redistributed to the cytoplasm in 79% of cells infected with the DA1 virus but remained cytoplasmic in 92% of cells infected with the TM598 virus. (B) HeLa cells infected with viruses DA1 and TM598 for 10 h were fixed and stained for the nuclear SC-35 splicing factor (left panels) and for viral antigen by using an anti-2A rabbit polyclonal antibody (right panels). White arrows point to cells positive for viral antigen. SC-35 staining was unaffected by viral infection.

FIG. 5.

Redistribution of PTB. L929 cells were left uninfected (NI) or were infected with KJ6 or TM659. At 4 h, 5 h 30 min, and 7 h after infection, cells were labeled for endogenous PTB. Viral antigen labeling done 7 h postinfection (hpi) showed that more than 98% of cells were infected in this experiment. Histograms present the means and standard errors of the means of 150 cell counts from triplicate infection experiments (B). Representative fields of PTB labeling are shown above (A). Note the conspicuous nuclear staining for uninfected cells and for cells infected for 4 h with TM659 and the extensive redistribution of PTB for cells infected with KJ6 at this time point. At 5 h 30 min and 7 h postinfection, several cells infected with TM659 displayed a punctate PTB cytoplasmic staining, often in addition to nuclear staining.