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Vijay Kumar, Ph.D. (vijay@icgeb.res.in) Scientist, Virology Group International Centre for Genetic Engg. and Biotechnology (ICGEB) New Delhi, India Virology-2015, Atlanta The HBx oncoprotein of hepatitis B virus promotes cell transformation by stimulating rDNA transcription and ribosome biogenesis 07 Dec. 2015
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HBV (DNA virus) Cell transformation in culture HBx transgenic mice Oncogenic properties of HBx Deregulation of cell cycle checkpoints Mitogenic signaling (e.g., MAPK, ras etc) Oncogenic cooperation (e.g., c-Myc) Interference with proteasomal machinery (e.g., Skp2, USP37) Possible mechanisms of HCC HBx oncoprotein 154 aa long, 16.5 KDa, Transactivator
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Does viral HBx support ribosome biogenesis during Hepatocarcinogenesis? Q Ribosome biogenesis: Achilles heel of cancer?
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Pol I transcription & ribosome biogenesis
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Nucleolar protein nucleophosmin (NPM) & ribosome biogenesis NPM NPM (B23), Nucleolin & fibrillarin ~20% of the total nucleolar protein content (~700 proteins)
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*p<0.001 Tahseen et al, Biomedica, 2006 o No. of AgNOR (Argyrophilic nucleolar organizer region) dots rises with increasing proliferative activity of cells Liver section with cirrhosis of the liver Liver section showing hepatocellular carcinoma
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Alteration in the nucleolar architecture of hepatocytes NormalHCC
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Ahuja et al., BBA, 1853, 1783-1795, 2015 Rajput et al., Virol J, 12, 62, 2015 How does HBx influence the UBF-NPM axis & enhance rDNA transcription, Ribosome biogenesis & Cell transformation Scope of our study
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Vector HBx α-UBF DAPI UBF expression in cells UBF expression in the presence of HBx
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Relative expression of UBF mRNA Huh7IHH Vector HBx * * * A B C HepG2.2.15 HepG2 A’ B’ C’ Vector HBx Huh7 α-UBF α-GAPDH α-HBx α-UBF α-GAPDH HepG2.2.15 α-HBx α-UBF α-GAPDH α-HBx IHH 1 1.8 Vector HBx 1 1.6 1 1.4 HepG2 UBF expression in the presence of HBx mRNA Protein
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A B Relative expression of UBF mRNA * * * ControlX15-mycControlX15-mycControlX15-myc Relative expression of UBF mRNA 1 3 6 Animal age (months) Control X15-myc Control X15-myc α-UBF α-GAPDH 1 3 6 Animal age (months) Control X15-myc 1 1.7 1 1.6 1 2.0 UBF expression in the HBx transgenic mouse liver mRNA Protein
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* * * * HBx + UBF UBF HBx Vector BrdU DAPI Merge BrdU incorporation in cells Cell proliferation following HBX-UBF co-expression
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Vector HBx UBF HBx + UBF * * Oncogenic co-operation between HBx & UBF Colony formation
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HBV HBx Upstream Binding Factor (UBF) RNA pol I on rDNA rDNA transcription & Ribosome biogenesis Cell proliferation & Cell transformation Summary : HBx-UBF interaction
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Do viruses alter Nucleolar protein levels? ?? Anna Greco, Rev. Med. Virol., 2009
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Status of Nucleolar proteins in the presence of HBx B D α-NPM α-Fibrillarin α-GAPDH 1 2 3 4 CT C T 1 3 Age (months) Tg. HBx mouse WB C HBx transfection Cell lines A Vector HBx Control HBx Tg mouse AgNOR staining: Cells Liver tissue 13% 23% DAPI α-Fibrillarin Merge Vector HBx
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NPM gene expression in the presence of HBx No change in NPM transcript levels (by RT-qPCR)
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NPM protein is stabilized in the presence of HBx NPM cleavage phosphorylation by CDK2 & proteolytic cleavage
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Direct interaction between HBx & NPM Coomassie staining -95- -70- -62- -51- -42- -29- MBP MBP-HBx His-NPM Marker MBP MBP-HBx Input His-Pull down MBP MBP-HBx Western Blot α-HBx α-NPM kDa 1 2 3 4 5 6 7 8 9 His-NPM + His-Pull down IB C -HBx -NPM IP: NPM V HBx Input IB A B
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A B HA-HBx FLAG-NPM DAPI Merge NPM C1 WT-NPM NPM C2 NPM C3 NPMc HepG2.2.15 HBx NPM DAPI Merge NPMNPM localization HBx localization Interaction with HBx WT-NPM (overexpressed / endogenous) Nucleoli Cytoplasm, nuclear, nucleolar + NPM C1 Nucleoli/ NucleoplasmCytoplasm- NPM C2 Nucleoli/ NucleoplasmCytoplasm- NPM C3 Nucleoli/ NucleoplasmCytoplasm- NPMcCytoplasm + Nucleolar translocatin of HBx by NPM
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HBx-NPM co-expression: rDNA transcription + HBx # # # * * A B
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HBx-NPM co-expression: Ribosome biogenesis & cell proliferation A B C
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HBx-NPM co-expression: Cell-transformation Vector HBx NPM+HBx NPM C3+HBx III Colony formation assay B A
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NPM Ribosome biogenesis Nucleosome Disassembly Nucleolus Nucleolar translocation rDNA promoter HBx Abnormal cell proliferation RNA Pol I Transcription factors Nucleosomes Caspase-3 HBx microenvironment o UBF expression o NPM stabilization o Nucleolar transport of HBx o rDNA transcription & cell proliferation Conclusions: Engagement of Pol I machinery by HBx - a novel mechanism of virus-induced oncogenesis. Summary: HBx-UBF-NPM axis in HCC HBx P NPM P HBx NPM cdk2 UBF expression stability
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Acknowledgements Richa Ahuja Pallavi Rajput Funding: ICGEB – Core grant CSIR - (Council of Scientific and Industrial Research, Govt. of India) DBT (Department of Biotechnology, Govt. of India)
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Thank you Wildflower Hall, Shimla, India
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