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. 2007 Apr 17;104(16):6684-9.
doi: 10.1073/pnas.0702158104. Epub 2007 Apr 11.

Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation

Kathrin Kindsmüller  1 , Peter Groitl, Barbara Härtl, Paola Blanchette, Joachim Hauber, Thomas Dobner
Affiliations

Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation

Kathrin Kindsmüller et al. Proc Natl Acad Sci U S A. .

Abstract

We have investigated the requirements for CRM1-mediated nuclear export and SUMO1 conjugation of the adenovirus E1B-55K protein during productive infection. Our data show that CRM1 is the major export receptor for E1B-55K in infected cells. Functional inactivation of the E1B-55K CRM1-dependent nuclear export signal (NES) or leptomycin B treatment causes an almost complete redistribution of the viral protein from the cytoplasm to the nucleus and its accumulation at the periphery of the viral replication centers. Interestingly, however, this nuclear restriction imposed on the wild type and the NES mutant protein is fully compensated by concurrent inactivation of the adjacent SUMO1 conjugation site. Moreover, the same mutation fully reverses defects of the NES mutant in the nucleocytoplasmic transport of Mre11 and proteasomal degradation of p53. These results show that nuclear export of E1B-55K in infected cells occurs via CRM1-dependent and -independent pathways and suggest that SUMO1 conjugation and deconjugation provide a molecular switch that commits E1B-55K to a CRM1-independent export pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effect of amino acid changes on stability and SUMOylation of E1B-55K mutant proteins. (A) Amino acid substitution mutations in E1B-55K mutant viruses. The leucine residues in the 55K NES known to be critical for CRM1-mediated nuclear export (11, 29) and the lysine residue that serves as the SUMO acceptor site (12) are indicated by triangles. Numbers refer to amino acid residues in the WT E1B-55K protein from H5pg4100. Amino acid changes in the E1B proteins from H5pm4101, H5pm4102, and H5pm4103 are indicated below. (B) Steady-state expression levels of E1B-55K proteins in WT and mutant virus-infected cells. A549 cells were infected with WT and mutant viruses at a multiplicity of 20 ffu per cell. Cells were harvested at the indicated times postinfection (p.i.), and total-cell extracts were prepared and subjected to immunoblotting by using anti-E1B-55K mouse monoclonal antibody (mab) 2A6. (C) Effect of mutations on SUMO1 conjugation of E1B-55K. Steady-state concentration of E1B-55K was detected by immunoblotting of total-cell lysates by using mab 2A6 (α-E1B; Left). The same extracts were subjected to immunoprecipitation with mab 2A6, and E1B/SUMO1 conjugates were visualized by SDS/PAGE and immunoblotting with anti-SUMO1 mouse mab 21C7 (α-SUMO1; Right). The bands representing the Ig heavy chain (IgH) and E1B-SUMO1 conjugate are indicated on Right.
Fig. 2.
Fig. 2.
Steady-state localization of E1B-55K and E2A-72K in WT and mutant virus-infected cells. (A) A549 cells were infected with WT and E1B mutant viruses at a multiplicity of 20 ffu per cell. Cells were fixed at 20 h p.i. and double-labeled in situ with anti-E2A-72K mouse mab B6–8 (α-DBP) and anti-E1B-55K rat mab 7C11 (α-E1B), and FITC- and Texas red-conjugated secondary antibodies, respectively. Representative anti-E2A (green; a, d, g, j, and m) and anti-E1B (red; b, e, h, k, and n) staining patterns are shown. The overlays of the green and red images are shown in c, f, i, l, and o (merge). In all panels, nuclei are indicated by a dotted line. (Magnification: ×7600.) (B) The boxed areas in g–i were enlarged 5-fold to illustrate the organization of the E1B NES mutant protein at defined sites around the periphery of the viral replication centers. (C) Subcellular distribution of WT and mutant E1B proteins in the presence of the CRM1 inhibitor LMB. A549 cells were infected at a multiplicity of 20 ffu per cell. At 21 h p.i., LMB was added to the medium. Three hours later, cells were fixed, and steady-state localization of E1B-55K was examined by indirect immunofluorescence as described above. In all panels, nuclei are indicated by a dotted line. (Magnification: ×7600.)
Fig. 3.
Fig. 3.
Effect of amino acid substitution mutations on steady-state localization of Mre11 and E1B-55K. A549 cells were mock-infected with WT or E1B mutant viruses at a multiplicity of 20 ffu per cell. Cells were fixed at 20 h p.i. and double-labeled in situ with anti-Mre11 mouse monoclonal antibody 12D7 (α-Mre11) and anti-E1B-55K rat mab 7C11 (α-E1B), and FITC- and Texas red-conjugated secondary antibodies, respectively. Representative anti-Mre11 (green; a, d, g, j, m, and p) and anti-E1B (red; b, e, h, k, n, and q) staining patterns are shown. The overlays of the green and red images are shown in c, f, i, l, o, and r (merge). Nuclei are indicated by a dotted line. Arrowheads in the merged images indicate Mre11 and/or E1B/Mre11-positive cytoplasmic inclusions. (Magnification: ×7600.)
Fig. 4.
Fig. 4.
Effect of amino acid substitutions on stability of p53 and Mre11. (A) Steady-state levels of Mre11 and p53 in WT and mutant virus-infected cells. A549 cells were infected with WT and E1B mutant viruses at a multiplicity of 20 ffu per cell. Cells were harvested at the indicated times p.i., and whole-cell extracts were prepared. Proteins from each time point were separated on SDS/PAGE and subjected to immunoblotting by using anti-Mre11 rabbit polyclonal antibody pNB-100–142, anti-p53 mouse mab DO-1, anti-E1B-55K mouse mab 2A6, and anti-E4orf6 rabbit polyclonal antibody 1807. (B) Coimmunoprecipitation of Mre11 with E1B-55K. Whole-cell extracts from infected A549 cells were prepared at 16 h after infection, coprecipitated (Ip) with mab 2A6 (α-E1B), and separated on SDS/PAGE followed by immunoblotting with antibody pNB-100–142.
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