Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

Jens Milbradt,Rike Webel,Sabrina Auerochs,Heinrich Sticht,Manfred Marschall

doi:10.1074/jbc.m109.063628

Jens Milbradt, Rike Webel + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.063628

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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.

Highlights

Human cytomegalovirus (HCMV)2 belongs to the ␤-herpesvirus subfamily, exhibiting worldwide distribution
Our study describes the formation of distinct punctate lamin A/C-depleted areas at the periphery of the nuclear envelope that can be induced by HCMV infection or by transient overexpression of individual nuclear egress complex (NEC) kinases
Indirect Immunofluorescence Double Staining and Confocal Laser Scanning Microscopy (CLSM)—HeLa cells or primary Human foreskin fibroblasts (HFFs) were grown on coverslips for transient transfection or HCMV infection, respectively

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructs—Expression plasmids coding for FLAG-tagged versions of pUL97 (i.e. pcDNA-UL97-FLAG, pcDNA-UL97(K355M)-FLAG, pcDNA-UL97-(1–595)-FLAG, and pcDNA-UL97-(181–707)-FLAG)), hemagglutinin-tagged pUL50 (pcDNA-UL50-HA), and PKC␣ fused to enhanced green fluorescent protein (EGFP; pEGFP-N1-PKC␣) were described previously [19, 21, 22, 27]. Indirect Immunofluorescence Double Staining and Confocal Laser Scanning Microscopy (CLSM)—HeLa cells or primary HFFs were grown on coverslips for transient transfection or HCMV infection, respectively. Three days post-infection, immunoprecipitation was performed as described previously [21] using 10 ␮l of anti-Pin pAb Coimmunoprecipitated samples and expression controls were subjected to standard Western blot analysis using anti-Pin pAb (A302-316A, recognizing epitope 113-163 of human Pin; BIOMOL) and anti-lamin A/C mAb. Bioinformatic Analysis—Candidates for functional protein interaction motifs in lamin A/C were identified in the ELM Motif Database [33] using the algorithm from Dinkel and Sticht [34]. Structural analysis and visualization were performed using the program DS ViewerPro (Accelrys Inc.)

RESULTS

Events by Phosphorylating Nuclear

DISCUSSION