Immunoprecipitation of MoMLV PICs from FLAG-tagged LEM protein-expressing cells. (A) FLAG-tagged LEM protein constructs. (B) Expression of the FLAG-tagged LEM proteins in NIH3T3 cells and immunoprecipitation of PICs from these cells. NIH3T3 cells were transfected with the expression vectors encoding FLAG-tagged proteins and, at 24 h after transfection, the cells were cocultured with MoMLV-producing cells. Cytoplasmic extracts from these cells were immunoprecipitated with an anti-FLAG monoclonal antibody. The weak band in lane 5 of panel B is background that is not reproducibly observed. Viral DNA was extracted from the captured immunocomplex and detected by Southern blotting (lower panel). Protein expression in the cocultured cells was analyzed by western blotting using anti-FLAG monoclonal antibody (upper panel). MW: molecular weight marker. Control experiments demonstrated that the anti-FLAG antibody immunoprecipitates each of the LAP2 isoforms with similar efficiency (Supplementary Figure S1).

Association of endogenous LAP2α with MoMLV PICs. Initial PICs (upper panels) and salt-stripped PICs (lower panels), made by coculture of NIH3T3 cells with MoMLV producer cells, were immunoprecipitated with the indicated antibodies and viral DNA was detected by Southern blotting.

In vitro association of LAP2α with MoMLV PICs. (A) PAGE of the purified GST-LAP2α fusion proteins. (B) GST pulldown assay of PICs. Cytoplasmic extract containg PICs was incubated with the indicated GST-LAP2α fusion proteins and precipitated by glutathione beads. The viral DNA in the bound fraction was detected by Southern blotting.

Stimulation of intermolecular integration of MoMLV PICs by LAP2α. Initial PICs (lanes 1–5) and salt-stripped PICs (lanes 6–10) were incubated with BAF or His-LAP2α and then added to the integration reaction mixture containing Φ × 174 RFI target DNA. After incubation, DNA products from the reaction were digested with BamHI and detected by Southern blotting. The 11.0-kb band results from intermolecular integration of the viral DNA into Φ × 174 RFI DNA (inter). The 5.6-kb band and the smear below it result from autointegration of the viral DNA into itself (auto). The 3.7- and 1.9-kb bands are the unreacted viral DNA containing 5′ LTR (L-end) or 3′ LTR (R-end), respectively (Lee and Craigie, 1994).

Stabilization of BAF/DNA complex by LAP2α in vitro. (A) Retention of intermolecular integration activity of MoMLV PICs after treatment with 400 mM KCl. The PIC fractions were incubated in the presence of 150 mM (lane 1), 400 mM (lane 2), or 750 mM (lane 3) KCl and, after gel filtration, samples were added to integration reaction mixtures containing Φ × 174 RFI DNA. Products were digested with BamHI and detected by Southern blotting. (B) Velocity sedimentation of BAF/DNA complexes with or without LAP2α. BAF and LAP2α were incubated with Φ × 174 DNA in buffer containing 115 mM KCl as indicated and then challenged with 150 or 400 mM KCl. After centrifugation in a sucrose gradient containing the same concentration of KCl, gradients were fractionated and the DNA was detected by Southern blotting.

Stabilization of the BAF/DNA complex by LAP2α requires both the LAP2 common and LAP2α-specific domains. Complexes of DNA, BAF and deletion derivatives of LAP2α were analyzed as in Figure 5B.

Depletion of LAP2α in vitro renders MoMLV PICs more sensitive to high salt. (A) Establishment of LAP2α-knockdown cells. NIH3T3 cells were transfected with a U6 promoter-driven siRNA expression vector encoding hairpin siRNA against LAP2α or noninteracting siRNA (siRNA control), and stable cell lines were selected. Cell lysate from each cell line was subjected to western blotting analysis using anti-LAP2 common domain (upper panel) or anti-lamins A/C (lower panel) monoclonal antibodies. MW: molecular weight markers. (B) Reduced levels of LAP2α are associated with MoMLV PICs from the LAP2α-knockdown cell line. The PIC fractions from each cell line were immunoprecipitated (IP, upper panel) with anti-LAP2α antibody and the recovered PICs were detected by Southern blotting. The lower panel shows the input PIC fractions without immunoprecipitation. (C) Diminished salt stability of PICs from the LAP2α-knockdown cells. The PIC fractions from each cell line were treated with the indicated concentration of KCl and, after gel filtration, assayed for integration activity. Although there was some quantitative variation between experiments, with residual intermolecular integration activity sometimes being observed after treatment of PICs from NIH3T3 cells with 750 mM KCl, PICs that derived the LAP2α-knockdown cells were consistently diminished in stability to 400 mM KCl.

Inhibition of MoMLV replication in LAP2α-knockdown cells. Each cell line was infected with MoMLV at low multiplicity for 2 h in the presence of polybrene and, after twice washing, cells were cultured in fresh culture medium. Culture supernatants were collected 3, 6, and 9 days after infection and virus spreading was monitored by RT activity assay.