Vijay Kumar, Ph.D. 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

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

Does viral HBx support ribosome biogenesis during Hepatocarcinogenesis? Q Ribosome biogenesis: Achilles heel of cancer?

Pol I transcription & ribosome biogenesis

Nucleolar protein nucleophosmin (NPM) & ribosome biogenesis NPM NPM (B23), Nucleolin & fibrillarin ~20% of the total nucleolar protein content (~700 proteins)

*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

Alteration in the nucleolar architecture of hepatocytes NormalHCC

Ahuja et al., BBA, 1853, , 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

Vector HBx α-UBF DAPI  UBF expression in cells UBF expression in the presence of HBx

Relative expression of UBF mRNA Huh7IHH Vector HBx * * * A B C HepG HepG2 A’ B’ C’ Vector HBx Huh7 α-UBF α-GAPDH α-HBx α-UBF α-GAPDH HepG α-HBx α-UBF α-GAPDH α-HBx IHH Vector HBx HepG2  UBF expression in the presence of HBx mRNA Protein

A B Relative expression of UBF mRNA * * * ControlX15-mycControlX15-mycControlX15-myc Relative expression of UBF mRNA Animal age (months) Control X15-myc Control X15-myc α-UBF α-GAPDH Animal age (months) Control X15-myc  UBF expression in the HBx transgenic mouse liver mRNA Protein

* * * * HBx + UBF UBF HBx Vector BrdU DAPI Merge  BrdU incorporation in cells  Cell proliferation following HBX-UBF co-expression

Vector HBx UBF HBx + UBF * * Oncogenic co-operation between HBx & UBF  Colony formation

HBV HBx Upstream Binding Factor (UBF) RNA pol I on rDNA rDNA transcription & Ribosome biogenesis Cell proliferation & Cell transformation Summary : HBx-UBF interaction

Do viruses alter Nucleolar protein levels? ?? Anna Greco, Rev. Med. Virol., 2009

Status of Nucleolar proteins in the presence of HBx B D α-NPM α-Fibrillarin α-GAPDH 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

NPM gene expression in the presence of HBx No change in NPM transcript levels (by RT-qPCR)

NPM protein is stabilized in the presence of HBx  NPM cleavage  phosphorylation by CDK2 &  proteolytic cleavage

Direct interaction between HBx & NPM Coomassie staining MBP MBP-HBx His-NPM Marker MBP MBP-HBx Input His-Pull down MBP MBP-HBx Western Blot α-HBx α-NPM kDa His-NPM + His-Pull down IB C  -HBx  -NPM IP: NPM V HBx Input IB A B

A B HA-HBx FLAG-NPM DAPI Merge NPM C1 WT-NPM NPM C2 NPM C3 NPMc HepG 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

HBx-NPM co-expression:  rDNA transcription + HBx # # # * * A B

HBx-NPM co-expression:  Ribosome biogenesis & cell proliferation A B C

HBx-NPM co-expression:  Cell-transformation Vector HBx NPM+HBx NPM C3+HBx III Colony formation assay B A

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

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)

Thank you Wildflower Hall, Shimla, India