Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus

Abstract

The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser(22) generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMV-infected cells.

FIGURE 1.

Induction of nuclear lamina-depleted areas in HCMV-infected and plasmid-transfected cells. A, HeLa cells were single (panels a–k and q–u) or cotransfected (panels l–p) with plasmids encoding tagged versions of pUL50, pUL97, or PKCα before cells were fixed and immunostained with the indicated antibodies 2 days post-transfection. DAPI, 4′,6-diamidino-2-phenylindole. C, HFFs were mock-infected (panels a–d) or infected with HCMV strain AD169 (panels e–h) or AD169-derived UL97 deletion virus expressing a GFP reporter (panels i–n). A subset of cells was treated with the inhibitor Gö6976 at 2 μm (panel n). At 3 days post-infection (AD169) or 3 weeks post-infection (AD169ΔUL97-GFP), cells were fixed and immunostained. Open arrowheads indicate lamin A/C-depleted areas. Inset images show enlargements of representative areas of the nuclear lamina. B and D, kinase-dependent nuclear lamin A/C alterations were quantitated in transfected (B) and HCMV-infected (D) cells in the presence or absence of Gö6976. Statistical significance compared with pUL97/pUL50 without Gö6976 treatment was calculated by Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

Phosphorylation of lamins A and C by pUL97 in vitro. 293T cells were cotransfected with catalytically active pUL97 (lanes 2–4, 5, 7, and 9), inactive C-terminally truncated pUL97 (lanes 6 and 8), or kinase-inactive point mutant K355M (lanes 10 and 11) together with constructs encoding lamin A or C fused to GFP. At 2 days post-transfection, cells were lysed and subjected to immunoprecipitation (IP) with the indicated antibodies, followed by in vitro kinase reaction with the precipitates. Labeled phosphorylation products were separated by SDS-PAGE/Western blot (Wb) transfer and visualized by exposure of the blots to autoradiography films (upper panels). Lysate control samples taken prior to immunoprecipitation were used for Western blot analysis with the indicated antibodies to monitor the levels of expressed proteins (lower panels). Control staining of the in vitro kinase assay (IVKA) blot using anti-GFP mAb confirmed the efficient precipitation of the substrate proteins (middle panels). RFP, red fluorescent protein; Phos., phosphorylated.

FIGURE 3.

pUL97/pUL50-induced reorganization of the nuclear lamina does not lead to free diffusion of coexpressed IE2p86. HeLa cells were cotransfected with combinations of constructs coding for pUL97, pUL50, and IE2p86 as indicated. Cells were fixed at 12 h post-transfection, immunostained with anti-lamin A/C mAb, and analyzed for IE2p86-GFP fluorescence. DAPI, 4′,6-diamidino-2-phenylindole; HA, hemagglutinin.

FIGURE 4.

Visualization of HCMV nuclear egress by tracking viral capsids by CLSM. HFFs were infected with HCMV TB40-UL32-EGFP. A and B, cells were fixed at various time points post-infection and immunostained with anti-lamin A/C mAb. A, monitoring of GFP-labeled capsids during the time course of infection. DAPI, 4′,6-diamidino-2-phenylindole. B, shown is the high resolution imaging of the nuclear egress of viral capsids (z stacks of two examples of HCMV-infected cells; panels f and g). Filled arrowheads indicate viral capsids; open arrowheads indicate lamina-depleted areas. C, shown is a time-lapse series of the movement of a GFP-labeled viral capsid at 3 days post-infection. At 62 hpi, live cell staining of the endoplasmic reticulum was performed (red). Filled arrowheads indicate the current position of the viral capsid; the dotted lines indicate the distance to the starting point of the viral capsid. Nu, nucleus; Cy, cytoplasm.

FIGURE 5.

A, model of the Pin1-lamin A interaction. The WW domain of Pin1 is shown in surface presentation and colored according to the electrostatic potential (red, most negative; blue, most positive). Ser¹⁸–Leu²⁴ of lamin A, which comprise the binding site, are shown in stick presentation and colored according to the atom type. The boxed region is shown as an enlargement below. B, detailed view of the interactions formed by phosphorylated Ser²². The phosphoryl group forms tight interactions with a serine (Ser¹⁶) and arginine (Arg¹⁷) of Pin1 (yellow dotted lines). C, the respective interactions cannot be formed by unphosphorylated Ser²².

FIGURE 6.

Interaction of Pin1 with lamin A in HCMV-infected cells. HFFs were infected with HCMV strain AD169 at m.o.i. = 0.1 and 1.0 or were left uninfected (mock). At 3 days post-infection, cells were lysed and used for co-immunoprecipitation (CoIP) analysis with anti-Pin1 pAb (A302-315A; lanes 1–3) or preimmune (pre) rabbit antiserum as a control (lane 4). Detection of co-immunoprecipitates (upper panels) and expression controls (lower panels) was performed on Western blots using anti-lamin A/C mAb or anti-Pin1 pAb (A302-315A), respectively. IP, immunoprecipitate.

FIGURE 7.

Subnuclear relocalization of Pin1 in HCMV-infected cells. HFFs were infected with HCMV AD169 and fixed at early (A) and late (B) time points post-infection for immunostaining with anti-Pin1 pAb (H-123), anti-lamin A/C mAb, and anti-UL44 mAb as indicated. HCMV-infected HFFs were treated with 2 μm Gö6976 (C). Open arrowheads indicate lamina-depleted areas. Inset images show enlargements of representative areas of the nuclear lamina. DAPI, 4′,6-diamidino-2-phenylindole.

FIGURE 8.

Hypothetical model of the course of events triggering the formation of nuclear lamina-depleted areas in HCMV-infected cells. ONM, outer nuclear membrane; phos., phosphorylation.