Epstein-Barr Virus BGLF4 Kinase Induces Disassembly of the Nuclear - PowerPoint PPT Presentation

Epstein-Barr Virus BGLF4 Kinase Induces Disassembly of the Nuclear Lamina to Facilitate Virion Production Chung-Pei Lee et al . Journal of Virology, 82 :11913-11926, 2008. Speaker: Shiang-Yuan Hsieh Advisor: Li-Kwan Chang, PhD Date: 2008.11.25 1

Epstein-Barr Virus � γ -herpesvirus human herpesvirus 4(HHV4) � Enveloped virus � Linear ds DNA, ~180 kb � Tissue tropism: B cells, T cells and epithelial cells 2 Dr. Chang LK ppt

Epstein-Barr Virus Life Cycle Latency proteins: Immediate-early proteins EBNAs Early proteins LMPs Late proteins Latency Stage Lytic Stage Spontaneous activation Stress(heat shock, UV) TPA/Sodium butyrate 3 Anti-IgG

Epstein-Barr Virus Associated Diseases � Direct: Infectious mononucleosis(IM) � Relative: Hodgkin’s disease(HD) Burkitt’s lymphoma(BL) Post transplant lymphoproliferative disorder (PTLD) Nasopharyngeal carcinoma(NPC) 4

EBV Lytic Cycle Transactivation Immediate-early genes Rta, Zta DNA replication Early genes BMRF1, BALF5, BALF2, BGLF4… etc Package, Release, membrane protein Late genes BFRF1, BFLF2… etc 5

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7 120 nm EBV nucleocapsid 38 nm ONM INM NPC

8 Envelopment Primary

The Cell: A Molecular Approach, 4e 9 INM ONM Nuclear Membrane NPC

Nuclear Lamina � A electro-dense meshwork lining the INM (nucleoplasmic face) � Composed by lamin A, B1, B2, C � Provide structural support for nuclear membrane 10 Aebi et al ., Naure. 1986

Nuclear Lamina � Bind to many INM proteins (emerin, lamin B receptor) � Bind to chromatin, DNA replication complex 11 Goldman et al ., Genes Dev. 2002

Lamin � Type-V intermediate filament � A type: lamin A, C( lmnA ) � nuclear stiffness B type: lamin B1, B2( lmnB1, B2 ) � fundamental structure of lamina nuclear shape Lamin B ONM Lamin A/C INM Head Rod Tail 12

Lamin assembly 13 The Cell: A Molecular Approach, 4e

Nuclear lamina disassembly in M phase Cdc2 (CDK1) The author assume that BGLF4 mimic Cdc2 function. 14 Molecular Biology of the Cell, 5e

EBV BGLF4 kinase � EBV early gene during lytic cycle � Ser/Thr protein kinase � Belong to the conserved family of herpesviral UL kinases � Substrate: - BGLF4, BMRF1, EBNA-2, EBNA-LP, Zta - EF-1 δ , condensin, topoisomerase II, MCM4 � BGLF4 phosphorylates EF-1 δ at Cdc2 target sites. Kawaguchi, Y. et al. (2003). J Virol 77 :2359-68 15

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Aims � Determine how EBV BGLF4 kinase regulates the dynamics of the nuclear lamina. � Explore the mechanism used by BGLF4 to regulate the egress of nucleocapsid. 17

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BGLF4 induces nuclear lamina disassembly?! 19

BGLF4 interacts with lamin A/C and induces nuclear lamina disassembly Kinase dead mutant IFA Co-IP (Immunofluorescence assay) (Co-immunoprecipitation assay) Immunofluorescence assay 20 HeLa cells (Confocal microscopy)

Cdc2 (CDK1) BGLF4 phosphorylates lamin A in vitro BGLF4 phosphorylates EF-1 δ at Cdc2 target sites. Kawaguchi, Y. et al. (2003). J Virol 77 :2359-68 21 HeLa cells Immunoprecipitation(IP) kinase assay

Ser-22,390,392 of lamin A are important for BGLF4-induced nuclear lamina disassembly IFA(Confocal) 22 HeLa cells

Ser-22,390,392 of lamin A are important for BGLF4-induced nuclear lamina disassembly 23

Conclusion(1/3) � BGLF4 phosphorylates lamin A/C and induces nuclear lamina disassembly. � Ser-22,390 and 392 of lamin A are important for BGLF4-induced nuclear lamina disassembly. Would nuclear lamina be disassembled by BGLF4 during EBV lytic cycle? 24

BGLF4 induces nuclear lamina disassembly during EBV reactivation EBV(+) EBV(-) 25

BGLF4 induces nuclear lamina disassembly during EBV reactivation Knockdown BGLF4!!! 26

Conclusion(2/3) � Nuclear lamina can be disassembled by BGLF4 during EBV reactivation. What are the effects of nuclear lamina disassembly on the EBV virion production? 27

Effects of nuclear lamina disassembly on the EBV virion production Immediate-early protein Early protein Late protein 28 EREV8 cells

Effects of nuclear lamina disassembly on the EBV virion production Late protein Early protein Late protein 29 EREV8 cells

EBV late protein BFRF1, BFLF2 Cytoplasm � EBV late gene ONM � Primary envelope proteins � BFRF1 � cytoplasm BFLF2 � nuclear Nucleo- capsid BFRF1 Primary INM BFLF2 Envelopment Nucleo- Nucleo- capsid capsid 30 Nucleus

Lamin A mutant reduce the expression of EBV late gene BFRF1/BFLF2 31 EREV8 cells

Lamin A mutant reduce virion production PCR 32 EREV8 cells

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