Nucleoporin 153 Arrests the Nuclear Import of Hepatitis B Virus Capsids in the Nuclear Basket (original) (raw)
Figure 2
Co-immune precipitations of human Nup153 and Nup153 fragments with different capsids.
A–D. Capsids were incubated with nuclear extract, GST-Nup153 or Nup153 fragments and precipitated with anti-capsid antibody-coated biomagnetic beads. The panels show immune blots after SDS PAGE. A–C: Detection by mAb414. A. 1 Immat-C, 2 Mat-C, 3 P-rC, 4 ΔC-rC. Co-precipitations of Nup153 from nuclear extracts showed that capsids precipitate Nup153 irrespective to the nucleic acid inside the capsid or the presence of the C terminus of the core protein. B. Co-precipitations of bacterially expressed GST-Nup153 showed that all capsid species directly interact with Nup153. 1 Immat-C, 2 Mat-C, 3 P-rC, 4 ΔC-rC. C. Specificity controls of GST-Nup153 co-precipitation exemplified by P-rC. 1. Positive control. P-rC + GST-Nup153 + anti capsid antibody-coated beads. 2. Without capsids + GST-Nup153 + anti capsid antibody-coated beads. 3. P-rC + without GST-Nup153 + anti capsid antibody-coated beads. D. Co-precipitation of GST-Nup153 fragments by P-rC. Immune detection of the Nup153 fragments by anti-GST antibodies bound to anti mouse coated biomagnetic beads. The GST-Nup153 fragment is given on the left of the panels, the capsid type at the top. p: GST-Nup153 fragment without precipitation (positive control). 1: P-rC + Nup153 fragment + anti capsid antibody-coated beads. 2: No capsids + Nup153 fragment + anti capsid antibody-coated beads. 3. P-rC + Nup153 fragment + uncoated beads. The figure shows that only the C1 fragment, which comprises aa 618–999 of Nup153, is precipitated by the capsids. E. Gel retardation of P-rC by GST-Nup153 N and C1 fragment on native agarose gels. The migration of GST-Nup153 N and C1 fragments without capsids is shown by anti-GST antibodies (right panel). The migration of the capsid without GST-Nup fragments is shown on the left panel using anti-capsid antibodies. The middle panel shows the capsid migration in the presence of GST-Nup153 N (left lane) and C1 (right lane). F. Gel retardation of P-rC by GST-Nup153 C2 fragment. Left: anti-capsid blot, right: anti-His blot. Left lanes: P-rC without Nup153 fragment, right lanes: P-rC with Nup153 fragment.