Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles

Abstract

The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein (Agno) has been shown to bind to HP1alpha and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1alpha from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.

Figure 1

Interaction of JCV Agno with HP1 in vivo. (A) Schematic representation of human HP1α and HP1γ showing the regions (bars) encoded by cDNA fragments isolated by a yeast two-hybrid assay with the N-terminal region of Agno as a bait. CD and CSD, chromo and chromo-shadow domains, respectively. (B) 293AG cells (HEK293 cells in which the expression of JCV Agno is inducible by Dox) were transfected with a vector for Myc-tagged human HP1α and were incubated in the absence or presence of Dox for 24 h. Cell lysates were subjected to immunoprecipitation with antibodies to Agno (anti-Agno), and the resulting precipitates were subjected to immunoblotting (IB) with anti-Myc. (C) Lysates prepared from 293AG cells after treatment with Dox for 48 h were subjected to immunoprecipitation with anti-HP1α or anti-Agno, and the resulting precipitates and cell lysates (Input) were subjected to immunoblotting with the same antibodies, as indicated. NMS, normal mouse serum; NRS, normal rabbit serum. (D) 293T cells were transfected with a vector for Agno and Myc-tagged human HP1α, β or γ, and were subjected to immunoprecipitation with anti-Agno. The resulting precipitates were subjected to immunoblotting with anti-Myc and anti-Agno.

Figure 2

Impaired ability of an Agno mutant to colocalize or interact with HP1. (A) Schematic representation of GST–EGFP fusion constructs of wild-type (WT) and mutant (C18) forms of Agno and EGFP fluorescence images showing their subcellular localizations in transfected HEK293 cells. (B) Lysates of 293T cells transiently expressing the WT or C18 forms of Agno were subjected to immunoprecipitation (IP) with anti-HP1α, and the resulting precipitates were subjected to immunoblotting (IB) with anti-Agno or anti-HP1α. (C) Immunofluorescence analysis of the expression of GST–EGFP-fused WT or C18 forms of Agno and of endogenous Lamin A/C in HEK293 cells. Enlarged dotted rectangles of the merged images are represented. Scale bars, 5 μm.

Figure 3

Agno-induced dissociation of the HP1α–LBR complex in vivo. (A) 293AG cells were incubated with Dox for the indicated times, after which cell lysates were subjected to immunoblotting with anti-Agno. Cells incubated with or without Dox for 24 h were also subjected to immunofluorescence analysis with anti-Agno. (B) 293AG cells were transfected with pEGFP-N1 or pLBR–EGFP together with pCMV–MycHP1α. The transfected cells were treated with Dox for 0, 3, 7 or 24 h. Cell lysates were then subjected to immunoprecipitation (IP) with anti-Agno or anti-EGFP, and the resulting precipitates were subjected to immunoblotting (IB) with anti-Myc. Cell lysates were also subjected directly to immunoblotting with anti-EGFP or anti-Myc.

Figure 4

Agno-induced increase in the lateral mobility of LBR. (A) Colocalization of LBR–EGFP detected by EGFP fluorescence and Agno detected with anti-Myc in 293AG cells. Arrows indicate an invagination of the NE in which LBR–EGFP and Agno are colocalized. Scale bar, 10 μm. (B) FRAP analysis of 293AG cells expressing LBR–EGFP and incubated in the absence or presence of Dox for 24 h. The fluorescence of LBR–EGFP in the boxed regions of the nuclear rim was irreversibly photobleached, and the recovery of fluorescence in these regions was monitored for 10 min. Representative images before (Pre) and at 1, 4, 7 and 10 min after bleaching are shown. Scale bars, 2 μm. (C) Quantification of the fluorescence recovery shown in (B). (D) Summary of fluorescence recovery ratios at 10 min after photobleaching. Each point represents an individual cell and the horizontal bars indicate the median values. The statistical significance of the difference between the two mean values was calculated by Student's two-tailed t-test.

Figure 5

Agno facilitates the nuclear egress of VLPs. The nuclei of Dox-treated (lower panels) or untreated (upper panels) 293AG cells were microinjected with VLPs and Cy3 (injection site marker; red fluorescence), and, after 1 h, the cells were subjected to immunofluorescence analysis with anti-VP1 (green). Blue lines indicate the rim of the nuclei and white lines indicate the edge of the cytoplasm.