Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen
- aInstitute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine
- bNational Research Institute of Chinese Medicine
- cInstitute of Microbiology, National Taiwan University College of Medicine
- dInstitute of Biochemistry and Molecular Biology, National Yang-Ming University School of Life Sciences, Taipei, Taiwan
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Fig 1Characterization of NESI protein. (A) Transient expression of NESI in Huh7 cells. Huh7 cells were transfected with plasmid pcDNA-NESI-V5HisTopo encoding an NESI protein with a V5His tag. Forty-eight hours posttransfection, cells were harvested and subjected to Western blot (WB) analysis with antibodies specific to the His tag. The NT represents nontransfected cells as a negative control. Arrows mark the major 55-kDa NESI-V5His protein and its modification forms. Molecular mass standards are shown in kDa on the left. (B) Glycosylation of NESI in Huh7 cells. Cell lysates prepared from Huh7 cells transiently expressing NESI-V5His proteins were subjected to immunoprecipitation with antibodies against the His tag. The immunoprecipitates were treated with PNGase F and Endo H as indicated, followed by Western blotting with the anti-His antibodies. The star indicates a nonspecific band. (C) NESI glycosylation in vitro. Plasmid pcDNA-NESI-V5HisTopo was subjected to an in vitro transcription/translation reaction in the absence or presence of canine pancreatic rough microsomes (RMs). The products were detected by Western blotting with the anti-V5 antibody following SDS-PAGE. The C represents a vector control analyzed in parallel. (D) Subcellular distribution of NESI. Cell lysates prepared from Huh7 cells transiently expressing NESI-V5His were fractionated into membrane and cytosol fractions. The fractions were subjected to immunoprecipitation and Western blot analysis with antibodies against the His tag. (E) Solubility of NESI in nonionic detergent. Huh7 cells transiently expressing the NESI-V5His proteins were lysed with PBST. The cell lysates were subjected to centrifugation to separate supernatant (Triton-soluble) and pellet (Triton-insoluble) fractions, followed by immunoprecipitation and Western blot analysis with antibodies against the His tag.
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Fig 2Identification of lamin A/C as an NESI-interacting protein. (A) Coimmunoprecipitation of cellular factors with the NESI-V5His protein. Cell lysates prepared from Huh7 cells transiently transfected with pcDNA-NESI-V5HisTopo encoding an NESI protein with a V5His tag (NESI-V5His) were subjected to a coimmunoprecipitation assay with the anti-V5 antibody and the control preimmune serum. Silver staining is shown. The stars mark the bands of cellular protein 1 and 2 that were coimmunoprecipitated with the NESI-V5His protein. Molecular mass markers are indicated on the left. (B) Coimmunoprecipitation of lamin A/C with NESI-V5His. Cell lysates prepared from Huh7 cells transiently transfected with pcDNA-NESI-V5HisTopo (NESI-V5His) or with pcDNA3.1 vector (Control) were subjected to a coimmunoprecipitation (IP) assay with anti-V5 antibodies and Western blot analysis with anti-lamin A/C (top) and anti-His tag (bottom) antibodies. (C and D) GST pulldown assays with GST-NESI fusion proteins and Huh7 cell lysates. A GST proteins consisting of truncated NESI proteins (indicated at the top by amino acid regions) bound to glutathione-Sepharose 4B beads were incubated independently with Huh7 cell lysates. Following the GST pulldown reaction, Western blot analysis was performed with antibodies specific to lamin A/C (top) and GST (bottom).
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Fig 3A direct interaction between NESI(1–185) and lamin A/C tail 2 domain. (A) Schematic representation of lamin A and its subdomains designated head, rod 1, rod 2, tail 1, and tail 2. (B and C) GST pulldown assay with the NESI protein and GST fusion proteins containing various domains of the lamin A (indicated at the top by amino acid regions). GST pulldown assays were performed with GST-lamin A fusion proteins precoupled to glutathione-Sepharose beads and lysates prepared from Huh7 cells expressing NESI-V5His (B) or purified His-NESI(1–185) protein (C). Following the GST pulldown reaction, Western blot analysis was performed with antibodies against the His tag of the NESI-V5His and His-NESI(1–185) proteins (top). GST fusion proteins that serve as loading controls were detected by Coomassie blue staining (bottom). Positions of molecular mass markers are indicated on the left.
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Fig 4Colocalization of NESI with lamin A/C and nucleoporins in Huh7 cells. Huh7 cells transiently expressing NESI-V5His were subjected to immunofluorescence staining with antibodies against the His tag followed by Alexa 488-conjugated goat IgG (green) and with anti-lamin A/C and anti-nucleoporins antibodies followed by Alexa 594-conjugated goat IgG (red) under a Leica TCS SP2 confocal microscope. Merged signals appear in yellow. In the top panel, Hoechst 33258 was used along with the secondary antibody to detect the nucleus.
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Fig 5NESI forms complexes with nucleoporins. (A) GST pulldown assay. GST pulldown assays were performed with Huh7 cell lysates and GST fusion proteins containing various domains of the NESI protein, indicated at the top, followed by Western blotting with antibodies specific to nucleoporins (top) and GST (bottom). (B) Coimmunoprecipitation analysis. Cell lysates prepared from Huh7 cells transiently expressing the NESI-V5His protein were subjected to immunoprecipitation assay with anti-His antibodies followed by Western blotting with antibodies specific to nucleoporins (top) and the V5 tag of NESI-V5His protein (bottom). NT, nontransfected cells used as negative controls.
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Fig 6Interactions of HDAg-L with NESI, lamin A/C, and nucleoporins. (A) GST pulldown assay. Various GST-NESI fusion proteins and a GST control protein bound to glutathione-Sepharose 4B beads were incubated with the cell lysates prepared from Huh7 cells transiently expressing HDAg-L. Following the GST pulldown reaction, Western blot analysis was performed with antibodies specific to HDAg (top) and GST (bottom). (B) Pro-205 is critical for HDAg-L to interact with NESI. GST pulldown assays were performed with GST-NESI(310–467) and total lysates prepared from Huh7 cells expressing wild-type (WT) HDAg-L or its mutants, HDAg-L(L199A), HDAg-L(D203A), and HDAg-L(P205A), as indicated, followed by Western blotting with antibodies against HDAg-L (top) and GST (bottom). (C to E) Association of HDAg-L with lamin A/C and nucleoporins. Huh7 cells were transfected with plasmids pECE-d-BE, pECE-d-BE(P205A), and pECE-d-SM encoding HDAg-L, HDAg-L(P205A), and HDAg-S, respectively, as indicated. At 2 days posttransfection, cells were harvested and subjected to immunoprecipitation with antibodies specific to lamin A/C (C and D) and nucleoporins (E), followed by Western blotting with antibodies against lamin A/C, nucleoporins, and HDAgs. The star marks the signal of the light chain.
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Fig 7Interference of HDAg-L nuclear export and HDV assembly in cells with downregulated lamin A/C expression. (A) shRNA knockdown of lamin A/C. Huh7 cells transfected with the plasmid pLKO.1-shLam expressing shRNA specific to lamin A/C were harvested at 2 days posttransfection for Western blot analysis with antibodies against lamin A/C. Cells without transfection (−) and cells expressing shRNA against luciferase served as controls. GAPDH was used as an internal control. (B) Effects of lamin A/C knockdown on the nuclear export of HDAg-L. Huh7 cells were cotransfected with plasmids pECE-d-BE, pECE-C-ES, and pLKO.1-shLam or pLKO.1-shLuc expressing HDAg-L, small HBsAg, and the shRNAs specific to lamin A/C or to luciferase, respectively. At 3 days posttransfection, cells were subjected to immunofluorescence staining with antibodies against HDAg. Three representative staining patterns of the HDAgs are shown: type I, nucleolus distribution; type II, both nucleolus and nucleoplasm distribution; type III, nucleolus, nucleoplasm, and cytoplasm distribution. For statistic analysis, fields containing at least 200 HDAg-positive cells each were randomly selected. Cell numbers bearing each type of the defined staining patterns of HDAg were counted and plotted as the percentage of the total number of the HDAg-positive cells. (C) Effects of lamin A/C knockdown on HDV assembly. Huh7 cells were cotransfected with plasmids expressing HDAg-L, small HBsAg, and shRNAs as described in the legend to panel B. Cells without coexpressing shRNA (−) served as a control. At 4 days posttransfection, VLPs released into the culture medium were harvested for Western blot analysis with antibodies specific to HDAg (top) and HBsAg (bottom). HDAg-L present in cell lysates (5% Input) was used as a control. NT, nontransfected cells.
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Fig 8A schematic diagram for the interacting domains of the NESI protein with lamin A/C, nucleoporin 153 (Nup153), and HDAg-L that bears a proline-rich NES. The putative glycosylation sites and transmembrane domains of the NESI protein are indicated.
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