Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus

Abstract

Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.

FIG. 1.

Intracellular localization of endogenous cellular proteins in uninfected and rhinovirus-infected cells. (A) Uninfected cells (mock infected) or HeLa cells infected with rhinovirus for 6 h were fixed and stained with an antibody directed against nucleolin. In the FITC panels, cells were examined with an FITC filter to detect the indicated antibodies. In the DNA panels, the same field was examined with a UV filter to visualize Hoechst staining of nuclei. (B) Visualization of La was performed as described for panel A. (C) Visualization of Sam68 was performed as described for panel A.

FIG. 2.

Intracellular localization of EGFP and EGFP-NLS molecules in uninfected and rhinovirus-infected cells. (A) HeLa cells stably expressing EGFP-NLS were mock infected or infected with rhinovirus as indicated. Cells were processed and examined by fluorescent microscopy 6 h after infection. EGFP fluorescence was visualized with an FITC filter. In the DNA panels, Hoechst-stained nuclei were examined with a UV filter. The panels on the right show the FITC and Hoechst images merged. (B) HeLa cells stably expressing EGFP fusion proteins were examined as described for panel A.

FIG. 3.

Intracellular localization of EGFP-M9NLS and EGFP-M9G₂₇₄A molecules in mock-infected and rhinovirus-infected cells. (A) HeLa cells were transiently transfected with plasmid pEGFP-M9NLS (EGFP-M9) and mock infected or infected with rhinovirus at 40 h after transfection and examined 6 h after infection. Labeling of panels is as described in the legend to Fig. 2. (B) Same as panel A, except cells were transfected with plasmid pEGFP-M9G₂₇₄A (EGFP-M9MT).

FIG. 4.

Intracellular localization of cellular proteins in uninfected and rhinovirus-infected cells. (A) Uninfected cells (mock infected) or HeLa cells infected with rhinovirus for 6 h were fixed and stained with an antibody directed against hnRNP A1. In the FITC panels, cells were examined with an FITC filter to detect indicated antibodies. In the DNA panels, the same field was examined with a UV filter to visualize Hoechst staining of nuclei. (B) Visualization of hnRNP K was performed as described for panel A. (C) Visualization of hnRNP C was performed as described for panel A. (D) Visualization of SC35 was performed as described for panel A.

FIG. 5.

Cell-free nuclear import assays. (A) Uninfected cells (mock infected) or cells that had been infected with rhinovirus for 6 h were permeabilized and used in an in vitro nuclear import assay. Assays were carried out in the presence (+RRL) or absence (−RRL) of RRL as a source of cytosolic factors. Top panels show GFP with an FITC filter, and bottom panels show Hoechst staining of DNA with a UV filter. All GFP images were acquired in Photoshop 5.0 (Adobe Systems, Inc.) with identical exposure times and manipulations. (B) Same as panel A, except creatine kinase, creatine phosphate, ATP, and GTP were omitted from the reaction. GFP images were acquired with the same exposure time as panel A.

FIG. 6.

Analysis of nuclear pore complex composition in rhinovirus-infected HeLa cells. (A) Fifty micrograms of whole-cell lysates prepared from mock-infected cells or cells that had been infected with rhinovirus for the indicated length of time was analyzed by immunoblotting with monoclonal antibody 414 to detect Nup153 and p62 or MS3 to detect nucleolin. eIF4GI was detected with rabbit polyclonal sera. An asterisk indicates rhinovirus-specific degradation products of eIF4GI. hpi, hours postinfection. (B) Indirect immunofluorescence with monoclonal antibodies 414 and SC-7292 to detect nucleoporins and lamins, respectively. Cells were either uninfected (mock infected) or infected with rhinovirus for 6 h (rhinovirus infected). The top panels show cells examined with an FITC filter, and the bottom panels show the same fields examined with a UV filter to detect Hoechst staining. FITC images for a given antibody were acquired with identical exposure times and adjustments.

FIG. 7.

Analysis of Nup153 and p62 in apoptotic and picornavirus-infected cells. (A) Electrophoresis of genomic DNA isolated from HeLa cells following various treatments. mock, lysate from mock-infected cells; polio, lysates from cells that have been infected with poliovirus for 5 h; rhino, lysates from cells that have been infected with rhinovirus for 6 h; ActD/Cx, lysates from cells that have been treated with actinomycin D and cycloheximide for 6 h; z-VAD, z-VAD-fmk added immediately following virus adsorption or coincident with the addition of actinomycin D and cycloheximide; M, molecular size markers (kilobases). (B) Fifty micrograms of whole-cell lysates prepared from cells treated as described in panel A was analyzed by immunoblotting to detect PARP. The positions of the full-length and cleaved forms of PARP are indicated by PARP and PARP∗, respectively. The positions of molecular mass markers (in kilodaltons) are indicated. (C) Fifty micrograms of whole-cell lysates was analyzed by immunoblotting with monoclonal antibody 414 to detect Nup153 and p62. The positions of the full-length and cleaved forms of Nup153 are indicated by Nup153 and Nup153∗, respectively. The positions of molecular mass markers (in kilodaltons) are indicated.