1. Hepatitis B Virus

2. Historical Background • Only human and chimpanzee-parenteraly • S protein of HBV 1963 B.S. blumberg • D.S. Dane – 42nm endogenous DNA polymerase activity 4. 1979 – cloning and sequencing S, C, P, X 5. Chronic hepatitis, hepatocellular carcinoma 6. Noncytocidal infection of hepatocyte

9. Hepatotropism • level of viral gene expression • Reservoir for virus to persist • Phagocytic activity detection of viral nucleic acid • Mediated by pre S1 • HBsAg filaments (rich in LHBs), 20nm particle (mainly SHBs) • Inhibition of binding with Mab (27-49 AAs) • Pre S2 supportive function Cellular receptor membrane -bound interleukin-6 SHBs -Apolipoprotein H, endonexin II  no signal receptor

10. Viral Genome • Relaxed circular, partially duplex DNA • P protein : covalently binding to 5' end of minus strand • 2 direct repeat (DR1, DR2) • 4 ORF ( C, P, PreS/S, X )

12. Viral replication HBV life cycle

13. Viral uptake Long-term cultivated hepatocytes • not susceptible but productive • Primary hepatocyte culture • Increase copy number of intercellular viral DNA • Endocytotic pathway (DHBV uptake) no data for HBV • Susceptibility with protease (HepG2) pre S2 domain

16. Nuclear entry of the viral genomes • Core particle in the nuclear (30nm 34nm diameter) – upper limit of nuclearpore 30nm • Nuclear localization signal of the carboxyl terminal of the core molecule ( fig. ) • Transfected cell – 1/1000 nuclear DNA • Viral polymerase – mediated transport of the viral genome • Amino- or/and carboxyl truncated pol – much more efficient

18. Formation of covalently closed circular (ccc) DNA • cccDNA is the template for the transcription of viral mRNA • cccDNA detected with in the first 24h • Prior to cccDNA • Removal of the covalently bound polymerase • Removal of RNA primer • Completion of (+)DNA strand • Repair and ligation of the gaps – maybe cellular DNA polymerase is essential  Core could also act as a vector for the nuclear transport of viral DNA  cccDNA accumulates only in nuclears

19. Synthesis of RNAs and proteins • 4 promoters and 2 enhancers, liver-enriched transcription factor at least 5 RNA • 5’-cap 3’- polyA (TATAAA) • Common 3’ end seem to be unspliced • X, preS1/S2/S, C/Pol

21. Transcription factor

23. X protein • Dispensable for infection, viral replication efficient infection in vivo, potential oncogenicity • The function of X cloning a natural viral infection is not known • Dispensable for viral DNA synthesis in infected tissue culture cells • Activate the transcription of host genes (MHC, C-myc)and viral genes • Stabilizes viral RNAs, not a DNA binding protein • Clear that X-defective virus is unable to initiate infection in vivo

25. S protein • 2.1 Kb, does not contain a sharply defined 5’ end, well transcribed in a variety of different cell lines • LHBS - 2.4Kb, transcribe in a tissue-dependent manner • HNF1 – liver specific factor is essential for virus secretion • Three s-glycosylated, type II transmembrane proteins, multimer by disulfide • Dane particle – L and M (equal amount, 30%) • Filamentous and spherical particles – 100-fold excess (~100g/ml) • Ab to S protein is enough • preS1 domain – viral receptor • preS2 – not essential for virus infection

28. Core protein • One core/e promoter, tissue dependent • 30bp longer – preC sequence • preC – hydrophobic -helix (signal) • Arginine center (C-termus) - bind NA • Maybe involved in 1st and 2nd DNA synthesis translation initiation of pol - internal entry or leaky scanning • Core particle – 36nm

32. polymerase • TP, spacer, pol, RNase H • Conserved motif – A~E (YMDD) • TP – critical role in the packaging of pg RNA • Cellular factor – HSP90, HSP70, HSP60

34. TP

35. Genome replication

36. Assembly of the nucleocapsid • Assembled spontaneously nonspecific incapsidation of RNA • C-terminal is not sufficient to mediate the specific encapsidation • Polymerase is required for specific RNA packing • -sequence, cellular factor -RNA + pol  conformation change -RNA + pol  core • Protein kinase C activity

39. Re-entry of mature core particle • New infection • Re-entry : amplification of cccDNA up to 50 copies : mutant DHBV(defective for S protein) 50 fold over • Regulation via envelopment

40. Envelopment and secretion of HBV • S proteins : rough ER • Virion formation depends on viral DNA synthesis • Protein maybe phosphorylation of the DHBV core protein • Binding of cores to the N-terminal of S  budding of the core particle across the ER membrane • Glycosylation of Asparagine of S  blood stream

42. Transient infection -1-6 months -e antigen : neonatal tolerogen -decrease hepatocyte half life -> hepatocyte replacement for a short period Chronic infection -high level HBsAg -10-15% of chronic infection -> Cirrhosis, liver cancer -treatment : lamivudine ( inhibition of p protein ) after 1 year, lamivudine resistance virus : a interferon

43. Liver Cancer Direct model - X protein ( Transgenic mouse ) - Truncated M -> induce integration to host chormosome (rarely) - Enhancer II -> activation N-myc ( only WHV, not HBV ) - p53 mutation ( 249 codon G->T ) of 80-90% HCC patient : multiple step - Activation of insulin-like growth factor(IGFII) ( only WHV )Indirect model - HBV infection ( L pretein overexpression )-> liver cell injury