FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2030706868> ?p ?o ?g. }

• W2030706868 endingPage "13989" @default.
• W2030706868 startingPage "13979" @default.
• W2030706868 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 Ser22 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. 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 Ser22 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. Human cytomegalovirus (HCMV) 2The abbreviations used are: HCMVhuman cytomegalovirusINMinner nuclear membranePKCprotein kinase CNECnuclear egress complexPPIasepeptidyl-prolyl cis/trans-isomeraseEGFPenhanced green fluorescent proteinHFFshuman foreskin fibroblastsm.o.i.multiplicity of infectionCLSMconfocal laser scanning microscopypAbpolyclonal antibodymAbmonoclonal antibodyhpihours post-infection. belongs to the β-herpesvirus subfamily, exhibiting worldwide distribution. When infecting immunocompetent individuals, HCMV possesses low pathogenicity, causing mainly asymptomatic infections. In immunocompromised or immunosuppressed hosts, HCMV infection can cause severe and even life-threatening diseases, including pneumonitis, retinitis, hepatitis, encephalitis, and gastroenteritis (1Mocarski E.S. Shenk T. Pass R.F. Knipe D.M. Howley P.M. Fields Virology. Lippincott/Williams & Wilkins, Philadelphia, PA2007: 2701-2772Google Scholar, 2Vancíková Z. Dvorák P. Curr. Drug Targets Immune Endocr. Metab. Disord. 2001; 1: 179-187Crossref PubMed Scopus (107) Google Scholar, 3Drew W.L. Clin. Microbiol. Rev. 1992; 5: 204-210Crossref PubMed Scopus (27) Google Scholar). human cytomegalovirus inner nuclear membrane protein kinase C nuclear egress complex peptidyl-prolyl cis/trans-isomerase enhanced green fluorescent protein human foreskin fibroblasts multiplicity of infection confocal laser scanning microscopy polyclonal antibody monoclonal antibody hours post-infection. HCMV replication is based on a nuclear phase, a characteristic of most DNA viruses. Transition from the nuclear to the cytoplasmic phase is determined by the nuclear exit of DNA-filled capsids budding through the inner nuclear membrane (INM) (4Mettenleiter T.C. Klupp B.G. Granzow H. Virus Res. 2009; 143: 222-234Crossref PubMed Scopus (302) Google Scholar, 5Sanchez V. Spector D.H. Science. 2002; 297: 778-779Crossref PubMed Scopus (17) Google Scholar, 6Sampaio K.L. Cavignac Y. Stierhof Y.D. Sinzger C. J. Virol. 2005; 79: 2754-2767Crossref PubMed Scopus (111) Google Scholar). The site-specific budding of viral capsids through distinct locally occurring invaginations in the INM of HCMV-infected cells was clearly illustrated by Buser et al. (7Buser C. Walther P. Mertens T. Michel D. J. Virol. 2007; 81: 3042-3048Crossref PubMed Scopus (62) Google Scholar) using electron microscopic analysis. The proteinaceous network of the nuclear lamina, underlying the INM, constitutes a major obstacle for the nuclear egress of capsids. Lamins, belonging to type V intermediate filament proteins, are the main constituents of the nuclear lamina and are grouped into A and B types. A-type lamins (A, C, AΔ10, and C2; collectively lamin A/C) result from alternative splicing of the LMNA gene. B-type lamins are encoded by the LMNB1 (B1) or LMNB2 (B2, B3) gene (8Goldman R.D. Gruenbaum Y. Moir R.D. Shumaker D.K. Spann T.P. Genes Dev. 2002; 16: 533-547Crossref PubMed Scopus (493) Google Scholar, 9Gruenbaum Y. Margalit A. Goldman R.D. Shumaker D.K. Wilson K.L. Nat. Rev. Mol. Cell Bio. 2005; 6: 21-31Crossref PubMed Scopus (689) Google Scholar). A major function of the nuclear lamina is to maintain the structure of the nuclear envelope. During mitosis, the nuclear lamina has to be transiently disassembled. This dynamic process is regulated by destabilizing phosphorylation of lamins at specific sites. In particular, it is well established that CDK1 (cyclin-dependent kinase 1; Cdc2) is mainly responsible for phosphorylation of lamins during mitosis (10Heald R. McKeon F. Cell. 1990; 61: 579-589Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 11Ward G.E. Kirschner M.W. Cell. 1990; 61: 561-577Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 12Peter M. Nakagawa J. Dorée M. Labbé J.C. Nigg E.A. Cell. 1990; 61: 591-602Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 13Likhacheva E.V. Bogachev S.S. Membr. Cell Biol. 2001; 14: 565-577PubMed Google Scholar). CDK1-dependent phosphorylation of lamin A/C occurs at Ser22, Ser392, and probably farther sites (10Heald R. McKeon F. Cell. 1990; 61: 579-589Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 14Hamirally S. Kamil J.P. Ndassa-Colday Y.M. Lin A.J. Jahng W.J. Baek M.C. Noton S. Silva L.A. Simpson-Holley M. Knipe D.M. Golan D.E. Marto J.A. Coen D.M. PLoS Pathog. 2009; 5: e1000275Crossref PubMed Scopus (165) Google Scholar, 15Peter M. Heitlinger E. Häner M. Aebi U. Nigg E.A. EMBO J. 1991; 10: 1535-1544Crossref PubMed Scopus (141) Google Scholar). HCMV blocks the cell cycle through the action of viral regulatory proteins (16Maul G.G. Negorev D. Med. Microbiol. Immunol. 2008; 197: 241-249Crossref PubMed Scopus (32) Google Scholar, 17Bain M. Sinclair J. Rev. Med. Virol. 2007; 17: 423-434Crossref PubMed Scopus (30) Google Scholar); however, it remains unclear whether HCMV is able to utilize the CDK1-based pathway for distortion of the nuclear lamina. A number of studies provided evidence that CMVs (including HCMV as well as murine CMV) and also other herpesviruses recruit another cellular lamin-phosphorylating kinase, namely protein kinase C (PKC). PKC is important for the phosphorylation and dissolution of the nuclear lamina (18Muranyi W. Haas J. Wagner M. Krohne G. Koszinowski U.H. Science. 2002; 297: 854-857Crossref PubMed Scopus (230) Google Scholar, 19Milbradt J. Auerochs S. Marschall M. J. Gen. Virol. 2007; 88: 2642-2650Crossref PubMed Scopus (85) Google Scholar). During mitosis of uninfected cells, there is substantial evidence that PKC is important for lamin phosphorylation and mitotic nuclear lamina disassembly (20Thompson L.J. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In HCMV-infected cells, the lamina-reorganizing activity of PKC appears to be supported by further lamin-phosphorylating protein kinases. The HCMV-encoded protein kinase pUL97 is recruited to the nuclear lamina (21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar, 22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and is highly important for efficient export of viral capsids to the cytoplasm (23Wolf D.G. Courcelle C.T. Prichard M.N. Mocarski E.S. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1895-1900Crossref PubMed Scopus (150) Google Scholar, 24Krosky P.M. Baek M.C. Coen D.M. J. Virol. 2003; 77: 905-914Crossref PubMed Scopus (198) Google Scholar). In particular, pUL97 phosphorylates lamins and has a destabilizing effect on the integrity of the nuclear lamina (22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). A recent study by Hamirally et al. (14Hamirally S. Kamil J.P. Ndassa-Colday Y.M. Lin A.J. Jahng W.J. Baek M.C. Noton S. Silva L.A. Simpson-Holley M. Knipe D.M. Golan D.E. Marto J.A. Coen D.M. PLoS Pathog. 2009; 5: e1000275Crossref PubMed Scopus (165) Google Scholar) confirmed the important regulatory role of pUL97 for nuclear egress by demonstrating a pUL97-dependent phosphorylation of A-type lamins at Ser22. This was causatively linked with morphological alterations of the nuclear lamina. On the basis of these observations, we postulated the formation of an HCMV-specific nuclear egress complex (NEC) composed of viral and cellular components (21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar). This complex may basically consist of pUL50, pUL53, pUL97, PKC, p32, and the lamin B receptor (21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar). In particular, the direct and indirect recruitment of PKC and pUL97, respectively, is mediated by viral pUL50. Whereas pUL50 is principally sufficient for PKC recruitment (21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar), other viral proteins such as pUL53 appear to enhance this function. pUL50 and pUL53 represent an interactor pair of essential nuclear egress proteins that are conserved among herpesviruses (5Sanchez V. Spector D.H. Science. 2002; 297: 778-779Crossref PubMed Scopus (17) Google Scholar, 18Muranyi W. Haas J. Wagner M. Krohne G. Koszinowski U.H. Science. 2002; 297: 854-857Crossref PubMed Scopus (230) Google Scholar, 19Milbradt J. Auerochs S. Marschall M. J. Gen. Virol. 2007; 88: 2642-2650Crossref PubMed Scopus (85) Google Scholar, 25Mettenleiter T.C. Virus Res. 2004; 106: 167-180Crossref PubMed Scopus (223) Google Scholar, 26Mettenleiter T.C. Vet. Microbiol. 2006; 113: 163-169Crossref PubMed Scopus (83) Google Scholar). Here, we provide novel information about the composition, function, and regulatory complexity of the NEC. In particular, 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 NEC kinases. Searching for the molecular mode of the phosphorylation-triggered process of lamina reorganization, we detected the involvement of the peptidyl-prolyl cis/trans-isomerase (PPIase) Pin1. These findings led us to propose a model that may explain the regulatory key features of the NEC and HCMV nuclear egress. 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 PKCα fused to enhanced green fluorescent protein (EGFP; pEGFP-N1-PKCα) were described previously (19Milbradt J. Auerochs S. Marschall M. J. Gen. Virol. 2007; 88: 2642-2650Crossref PubMed Scopus (85) Google Scholar, 21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar, 22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 27Thomas M. Rechter S. Milbradt J. Auerochs S. Müller R. Stamminger T. Marschall M. J. Gen. Virol. 2009; 90: 567-578Crossref PubMed Scopus (45) Google Scholar). Plasmid pHM990, coding for a fusion protein of IE2p86 and GFP, was kindly provided by Dr. N. Tavalai (Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg) (28Tavalai N. Papior P. Rechter S. Leis M. Stamminger T. J. Virol. 2006; 80: 8006-8018Crossref PubMed Scopus (186) Google Scholar). The plasmids pEGFP-N1-lamin A and pEGFP-N1-lamin C code for lamin A and C, respectively, which are fused to EGFP (kindly provided by Dr. J. Broers, Cardiovascular Research Institute, University of Maastricht, Maastricht, The Netherlands) (29Broers J.L. Kuijpers H.J. Ostlund C. Worman H.J. Endert J. Ramaekers F.C. Exp. Cell Res. 2005; 304: 582-592Crossref PubMed Scopus (76) Google Scholar). HeLa and 293T cells were cultivated and transfected as described previously (19Milbradt J. Auerochs S. Marschall M. J. Gen. Virol. 2007; 88: 2642-2650Crossref PubMed Scopus (85) Google Scholar, 21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar). Human foreskin fibroblasts (HFFs) were cultivated in minimal essential medium containing 7.5% fetal calf serum. HCMV infection experiments were performed at a multiplicity of infection (m.o.i.) of 1.0 (or lower as indicated for specific experiments) using the laboratory strain AD169, the UL97 deletion mutant BAC213 (22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), or the recombinant TB40-UL32-EGFP virus expressing GFP fused to the capsid-associated tegument protein pUL32 (pp150, kindly provided by Prof. C. Sinzger, Institute of Medical Virology, University of Tübingen, Tübingen, Germany) (6Sampaio K.L. Cavignac Y. Stierhof Y.D. Sinzger C. J. Virol. 2005; 79: 2754-2767Crossref PubMed Scopus (111) Google Scholar). Gö6976 is an inhibitor of serine/threonine protein kinases (particularly pUL97 and PKC) (30Marschall M. Stein-Gerlach M. Freitag M. Kupfer R. van den Bogaard M. Stamminger T. J. Gen. Virol. 2001; 82: 1439-1450Crossref PubMed Scopus (71) Google Scholar). AG490 (tyrphostin) is an inhibitor of tyrosine protein kinases (31Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-648Crossref PubMed Scopus (848) Google Scholar). Compounds were purchased from Calbiochem. Stock solutions were prepared in Me2SO, and aliquots were stored at −20 °C. HeLa cells or primary HFFs were grown on coverslips for transient transfection or HCMV infection, respectively. At the indicated time points, cells were fixed and permeabilized following indirect immunofluorescence staining as described (19Milbradt J. Auerochs S. Marschall M. J. Gen. Virol. 2007; 88: 2642-2650Crossref PubMed Scopus (85) Google Scholar, 21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar). The following polyclonal (pAb) and monoclonal (mAb) antibodies were used to detect cellular and viral proteins: anti-lamin A/C mAb (636) and anti-Pin1 pAb (H-123, raised against amino acids 41–163 of human Pin1) (Santa Cruz Biotechnology), anti-FLAG mAb (M2) and anti-FLAG pAb (Sigma), anti-hemagglutinin pAb (HA.11; HiSS Diagnostics GmbH), anti-GFP mAb (clone 7.1/13.1; Roche Applied Science), anti-UL97 pAb (kindly provided by Prof. D. Michel, University of Ulm, Ulm, Germany) (32Michel D. Pavić I. Zimmermann A. Haupt E. Wunderlich K. Heuschmid M. Mertens T. J. Virol. 1996; 70: 6340-6346Crossref PubMed Google Scholar), and anti-UL44 mAb (BS 510; kindly provided by Prof. B. Plachter, University of Mainz, Mainz, Germany). Secondary antibodies used for double staining were fluorescein isothiocyanate- and Cy3-conjugated (Dianova). Images were acquired using a Leica TCS SP5 confocal laser scanning microscope equipped with a 63× HCX PL APO CS oil immersion objective lens (Leica) and analyzed using LAS AF software (Leica). HFFs were seeded into 2-well chambered coverglass units with coverslip quality glass bottoms (Lab-Tek, Nunc) at a density of 1 × 105 cells/well. The next day, the cells were infected with recombinant TB40-UL32-EGFP virus at a m.o.i. of 1.0. 62 h post-infection (hpi), the cells were washed with Hanks' balanced salt solution with calcium and magnesium (Invitrogen) and then incubated with prewarmed staining solution for live cell endoplasmic reticulum labeling (ER-Tracker red dye, Invitrogen) at a concentration of 1 μm for 20 min at 37 °C and 5% CO2. After replacing the staining solution with fresh probe-free medium, the cells were examined using a TCS SP5 confocal laser scanning microscope. The intracellular trafficking velocity of viral particles was determined by using LAS AF Version 1.8.2 (build 1465, Leica). The kinase activity of FLAG-tagged pUL97 was determined in vitro (2.5 μCi of [γ-33P]ATP) after immunoprecipitation of the kinase and the putative substrate proteins lamin A-GFP and lamin C-GFP from lysates of transfected 293T cells. Immunoprecipitates were subsequently pelleted, washed, and subjected to in vitro kinase assay reaction as described previously (19Milbradt J. Auerochs S. Marschall M. J. Gen. Virol. 2007; 88: 2642-2650Crossref PubMed Scopus (85) Google Scholar, 21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar). Finally, samples were prepared for separation by 12.5% SDS-PAGE, followed by transfer to nitrocellulose membrane (A. Hartenstein) by Western blotting. Autoradiographic membranes were exposed to a phosphorimager plate and measured using a BAS-2000 phosphorimager (Fuji Film. Co., Tokyo, Japan). HFFs were seeded into cell culture flasks at a density of 3.6 × 106 cells/flask. The next day, cells were infected with HCMV strain AD169 at m.o.i. = 0.1 and 1.0. Three days post-infection, immunoprecipitation was performed as described previously (21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar) using 10 μl of anti-Pin1 pAb (A302-315A, recognizing epitope 50-100 of human Pin1; BIOMOL) or preimmune rabbit antiserum, respectively. Co-immunoprecipitated samples and expression controls were subjected to standard Western blot analysis using anti-Pin1 pAb (A302-316A, recognizing epitope 113-163 of human Pin1; BIOMOL) and anti-lamin A/C mAb. Candidates for functional protein interaction motifs in lamin A/C were identified in the ELM Motif Database (33Puntervoll P. Linding R. Gemünd C. Chabanis-Davidson S. Mattingsdal M. Cameron S. Martin D.M. Ausiello G. Brannetti B. Costantini A. Ferrè F. Maselli V. Via A. Cesareni G. Diella F. Superti-Furga G. Wyrwicz L. Ramu C. McGuigan C. Gudavalli R. Letunic I. Bork P. Rychlewski L. Küster B. Helmer-Citterich M. Hunter W.N. Aasland R. Gibson T.J. Nucleic Acids Res. 2003; 31: 3625-3630Crossref PubMed Scopus (519) Google Scholar) using the algorithm from Dinkel and Sticht (34Dinkel H. Sticht H. Bioinformatics. 2007; 23: 3297-3303Crossref PubMed Scopus (34) Google Scholar). The structure of lamin A in complex with Pin1 was modeled based on the known crystal structure of Pin1 in complex with a peptide from the RNA polymerase II C-terminal domain (Protein Data Bank code 1f8a) (35Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (426) Google Scholar). For this purpose, the C-terminal domain ligand sequence was replaced with that of lamin A using the lowest energy rotamers for the non-conserved amino acid side chains. The complex was subsequently refined by 100 steps of energy minimization using SYBYL 7.3 software (Tripos, L.P.). Structural analysis and visualization were performed using the program DS ViewerPro (Accelrys Inc.). Previous investigations demonstrated that HCMV replication exerts drastic morphological alterations on the nuclear lamina (21Milbradt J. Auerochs S. Sticht H. Marschall M. J. Gen. Virol. 2009; 90: 579-590Crossref PubMed Scopus (72) Google Scholar, 22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The effects described include a thinning of the lamina layer, loss of the typical rim staining of individual lamina components, internal nuclear speckling of lamins, and modified antigenic detectability (7Buser C. Walther P. Mertens T. Michel D. J. Virol. 2007; 81: 3042-3048Crossref PubMed Scopus (62) Google Scholar, 14Hamirally S. Kamil J.P. Ndassa-Colday Y.M. Lin A.J. Jahng W.J. Baek M.C. Noton S. Silva L.A. Simpson-Holley M. Knipe D.M. Golan D.E. Marto J.A. Coen D.M. PLoS Pathog. 2009; 5: e1000275Crossref PubMed Scopus (165) Google Scholar, 22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). We investigated the fine-structured morphological organization of the nuclear lamina by CLSM and visualized effects produced by the expression of the viral protein kinase pUL97 (supplemental Fig. S1A). In vector-transfected cells, the staining for lamin A/C showed a strict rim signal (supplemental Fig. S1A, panels a–e). As a striking result, those cells expressing a catalytically active version of pUL97 (full-length pUL97 or N-terminally truncated pUL97-(181–707)) showed an increase of lamin signals throughout the nuclear plasma (supplemental Fig. S1A, panels f–p). The rim staining changed to a more homogeneous nuclear distribution (supplemental Fig. S1A, panels k and p). No induction of similar effects was observed for catalytically inactive versions of pUL97 (point mutant pUL97(K355M) or C-terminally truncated pUL97-(1–595)) (22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) (supplemental Fig. S1A, panels q–z). To ensure comparable levels of pUL97 expression, semiquantitative Western blot analysis was performed (supplemental Fig. S1B). Interestingly, transient expression of pUL97 produced only a rather moderate phenotype of lamin A/C reorganization (as shown in supplemental Fig. S1), whereas coexpression of pUL97 and egress protein pUL50 potentiated the lamina-reorganizing activity (Fig. 1A). Coexpression of pUL50 and pUL97 led to punctate distortions at the periphery of the nuclear envelope, which clearly represented centers of massive lamin A/C reorganization (Fig. 1A, panels l–p). The potentiating effect of pUL50 might be explained by an indirect protein interaction bridged by the pUL97-interacting cellular adapter protein p32 (22Marschall M. Marzi A. aus dem Siepen P. Jochmann R. Kalmer M. Auerochs S. Lischka P. Leis M. Stamminger T. J. Biol. Chem. 2005; 280: 33357-33367Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Coexpression of pUL53 and pUL97 did not induce a lamina reorganization as demonstrated for pUL50 and pUL97 (data not shown). Using pUL50 only induced some minor lamina alterations restricted to a small fraction of cells; however, in no case was a local depletion of the nuclear lamina observed (Fig. 1A, panels f–k). Quantification of pUL50/pUL97-coexpressing cells revealed that 8.2% of cells staining positive for pUL50 showed distinct distortions of nuclear lamin A/C (Fig. 1B). Treatment with the kinase inhibitor Gö6976 significantly reduced this effect to 1.8%, confirming the central role of protein kinase activity. The indolocarbazole compound Gö6976 possesses strong inhibitory potential against pUL97 (30Marschall M. Stein-Gerlach M. Freitag M. Kupfer R. van den Bogaard M. Stamminger T. J. Gen. Virol. 2001; 82: 1439-1450Crossref PubMed Scopus (71) Google Scholar, 36Marschall M. Stein-Gerlach M. Freitag M. Kupfer R. van den Bogaard M. Stamminger T. J. Gen. Virol. 2002; 83: 1013-1023Crossref PubMed Scopus (73) Google Scholar) and an additional activity against PKC (37Goekjian P.G. Jirousek M.R. Curr. Med. Chem. 1999; 6: 877-903PubMed Google Scholar). The use of a kinase-inactive point mutant (K355M) led to a statistically significant difference in measurable lamina distortions compared with wild-type pUL97. However, a substantial portion of cells still showed measurable effects (4.4%) (Fig. 1B), suggesting that endogenous PKC activity, possibly associated with pUL97(K355M)-derived protein complexes, might also contribute to the alterations in lamin A/C. This notion was underscored by the finding that Gö6976 treatment markedly reduced the effect of mutant K355M (2.6%). The direct phosphorylation of lamins by pUL97 was analyzed by performing in vitro kinase assay. pUL97 and its putative substrates lamin A-GFP and lamin C-GFP were immunoprecipitated from lysates of transiently transfected cells. In the in vitro phosphorylation reaction, lamin A-GFP and lamin C-GFP were phosphorylated by pUL97 (Fig. 2, lanes 2, 3, 5, and 7). The vector control (Fig. 2, lane 4), red fluorescent protein (lane 1), or GFP (data not shown) did not produce a phosphorylation signal. An additional antibody control confirmed the specificity of the reaction (Fig. 2, lane 9). Efficient precipitation of the substrate proteins was confirmed by precipitation control staining (Fig. 2, middle panels). In accordance with the findings shown in supplemental Fig. S1, lamin phosphorylation was exclusively detectable for catalytically active pUL97 (N-terminally truncated pUL97-(181–707)) (Fig. 2, lanes 5 and 7), whereas an inactive C-terminally truncated version (pUL97-(1–595)) (lanes 6 and 8) or a kinase-inactive point mutant (pUL97(K355M)) (lanes 10 and 11) did not produce a phosphorylation signal. We addressed the question as to whether additional protein kinases are involved in lamina-modifying processes. Expression experiments with PKCα-GFP were performed to investigate whether PKC can induce morphological alteration of the nuclear lamina similarly to pUL97. In fact, the formation of distinct punctate distortions of nuclear lamin A/C was also detected in PKC-expressing cells (Fig. 1A, panels q–u). The inset magnification of the merged picture (Fig. 1A, panel u) shows PKCα-GFP mainly in a cytoplasmic localization adjacent to the regular nuclear rim shaped by lamin A/C. The nuclear rim is significantly broken so that no lamin signal remains detectable. Similar alterations were not detectable in control cells lacking PKCα-GFP expression (Fig. 1A, panel e). We monitored the induction of nuclear lamina alterations (lamin A/C) during HCMV replication and the localization of pUL97. In HCMV-infected primary HFFs, pUL97 was found mostly associated with viral replication centers at late time points of infection (60–120 hpi) as described previously (38Marschall M. Freitag M. Suchy P. Romaker D. Kupfer R. Hanke M. Stamminger T. Virology. 2003; 311: 60-71Crossref PubMed Scopus (100) Google Scholar). With increasing time, a number of cells also contained pUL97 in a perinuclear localization, which was at least in part associated with the nuclear envelope (Fig. 1C, panel f). Pronounced distortions of nuclear lamin A/C were increasingly induced in the late phase of viral replication (Fig. 1C, panel h). When using an AD169-derived UL97 deletion virus expressing a GFP reporter (AD169ΔUL97-GFP), limited distortions of the nuclear lamina were observed (Fig. 1C, panels i–m). This type of distortion was characterized by a limited thinning of the nuclear lamina (lamin A/C) and by smaller depletions appearing with lower quantity compared with parental HCMV AD169. Quantification of the data revealed that most of the lamina-specific effect could be attributed to the presence of pUL97 (Fig. 1D). The lack of viral pUL97 expression (AD169ΔUL97-GFP) and/or the inhibition of kinase activity (Gö6976) substantially reduced the effect of nuclear lamina depletion. Thus, the measurable inhibitory potency of Gö6976 in cells in" @default.
• W2030706868 created "2016-06-24" @default.
• W2030706868 creator A5004691572 @default.
• W2030706868 creator A5029180430 @default.
• W2030706868 creator A5040808433 @default.
• W2030706868 creator A5065393453 @default.
• W2030706868 creator A5079421880 @default.
• W2030706868 date "2010-04-01" @default.
• W2030706868 modified "2023-09-30" @default.
• W2030706868 title "Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus" @default.
• W2030706868 cites W1509261109 @default.
• W2030706868 cites W1557338439 @default.
• W2030706868 cites W1973836212 @default.
• W2030706868 cites W1975429375 @default.
• W2030706868 cites W1985880387 @default.
• W2030706868 cites W2009811254 @default.
• W2030706868 cites W2017132720 @default.
• W2030706868 cites W2020520526 @default.
• W2030706868 cites W2026696908 @default.
• W2030706868 cites W2031588422 @default.
• W2030706868 cites W2040541452 @default.
• W2030706868 cites W2043485308 @default.
• W2030706868 cites W2044267903 @default.
• W2030706868 cites W2050094053 @default.
• W2030706868 cites W2052817464 @default.
• W2030706868 cites W2056952591 @default.
• W2030706868 cites W2059023146 @default.
• W2030706868 cites W2062371417 @default.
• W2030706868 cites W2065634301 @default.
• W2030706868 cites W2081021921 @default.
• W2030706868 cites W2082607775 @default.
• W2030706868 cites W2090997741 @default.
• W2030706868 cites W2097253678 @default.
• W2030706868 cites W2098677586 @default.
• W2030706868 cites W2105788709 @default.
• W2030706868 cites W2112694870 @default.
• W2030706868 cites W2113221891 @default.
• W2030706868 cites W2123837005 @default.
• W2030706868 cites W2127048642 @default.
• W2030706868 cites W2137952286 @default.
• W2030706868 cites W2138240302 @default.
• W2030706868 cites W2142939796 @default.
• W2030706868 cites W2143722577 @default.
• W2030706868 cites W2146101141 @default.
• W2030706868 cites W2153568126 @default.
• W2030706868 cites W2160623917 @default.
• W2030706868 cites W2161779139 @default.
• W2030706868 cites W2166912626 @default.
• W2030706868 cites W2168514491 @default.
• W2030706868 cites W2168696636 @default.
• W2030706868 cites W2170689152 @default.
• W2030706868 cites W2171236242 @default.
• W2030706868 cites W2340807121 @default.
• W2030706868 cites W2980345380 @default.
• W2030706868 cites W954261861 @default.
• W2030706868 doi "https://doi.org/10.1074/jbc.m109.063628" @default.
• W2030706868 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2859560" @default.
• W2030706868 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20202933" @default.
• W2030706868 hasPublicationYear "2010" @default.
• W2030706868 type Work @default.
• W2030706868 sameAs 2030706868 @default.
• W2030706868 citedByCount "87" @default.
• W2030706868 countsByYear W20307068682012 @default.
• W2030706868 countsByYear W20307068682013 @default.
• W2030706868 countsByYear W20307068682014 @default.
• W2030706868 countsByYear W20307068682015 @default.
• W2030706868 countsByYear W20307068682016 @default.
• W2030706868 countsByYear W20307068682017 @default.
• W2030706868 countsByYear W20307068682018 @default.
• W2030706868 countsByYear W20307068682019 @default.
• W2030706868 countsByYear W20307068682020 @default.
• W2030706868 countsByYear W20307068682021 @default.
• W2030706868 countsByYear W20307068682022 @default.
• W2030706868 countsByYear W20307068682023 @default.
• W2030706868 crossrefType "journal-article" @default.
• W2030706868 hasAuthorship W2030706868A5004691572 @default.
• W2030706868 hasAuthorship W2030706868A5029180430 @default.
• W2030706868 hasAuthorship W2030706868A5040808433 @default.
• W2030706868 hasAuthorship W2030706868A5065393453 @default.
• W2030706868 hasAuthorship W2030706868A5079421880 @default.
• W2030706868 hasBestOaLocation W20307068681 @default.
• W2030706868 hasConcept C11960822 @default.
• W2030706868 hasConcept C159047783 @default.
• W2030706868 hasConcept C185592680 @default.
• W2030706868 hasConcept C190062978 @default.
• W2030706868 hasConcept C2522874641 @default.
• W2030706868 hasConcept C2776409557 @default.
• W2030706868 hasConcept C2777732132 @default.
• W2030706868 hasConcept C2779820661 @default.
• W2030706868 hasConcept C2780114586 @default.
• W2030706868 hasConcept C2780727368 @default.
• W2030706868 hasConcept C59157529 @default.
• W2030706868 hasConcept C86803240 @default.
• W2030706868 hasConcept C95444343 @default.
• W2030706868 hasConceptScore W2030706868C11960822 @default.
• W2030706868 hasConceptScore W2030706868C159047783 @default.
• W2030706868 hasConceptScore W2030706868C185592680 @default.
• W2030706868 hasConceptScore W2030706868C190062978 @default.

• next