HBV Replication

1. HBV Replication 2012. 04. 16 Lab of Molecular Genetics Presented by HeewonJeong

2. Overview of HBV replication

3. Necessary steps After infection • Viral capsid disassembly • Transport of RC-DNA into the nucleus • Conversion of RC-DNA into cccDNA - Thus far, it is not yet known where in the cells capsid disassembly occurs and how RC-DNA is delivered into the nucleus

4. Nuclear import of HBV genome • 3 different possibilities for nuclear entry in theory • Viral DNA is released from the nucleocapsid and deproteinized outside the nucleus, then genome is imported into the nucleus • Incoming virus loses surface proteins during entry, and complete core particles are transported into nucleus • Disassembly of core particles outside the nucleus and transport mediated either by covalently linked polymerase or by non-assembled core protein subunits attached to polymerase-DNA complex

5. Nuclear import of HBV genome • Data (Kann et al.) suggested role for the viral reverse transcriptase of HBV covalently attached to viral genome probably contains a hidden nuclear localization signal (NLS) • In vitro transport assay using wild-type and protein-free DNA Transport system with polymerase-DNA complex was efficient

6. Structure of RC-DNA • Distinct features • (-)-DNA strand is complete while (+)-strands is less than full-length • 5’ end of (-)-DNA is covalently linked to P protein • 5’ end of (+)-strand consists of an RNA oligonucleotide, derived from the pgRNA, which served as the primer for (+)-strand synthesis Ref. Nassal et al. HBV replication, W.J of gastroenterology, 2007

7. RC-DNA to cccDNA conversion • Upon infection, RC-DNA is converted, inside the host cell nucleus, into a plasmid-like covalently closed circular DNA (cccDNA) • For cccDNA formation, all modifications need to be removed, and both strands need to be covalently ligated

8. Fill-in gap in (+) DNA • Removal of P protein at 5’ end of (-) DNA • By removal of peptide or amino acid remaining after initial • proteolysis event OR • By nucleolytic cleavage of DNA segment carrying P protein OR • By cleavage of bond between priming Tyr residue and 5’ end (-) DNA nt • - Remove RNA primer at 5’ end of (+) DNA • Ligation of (-) and (+) strand DNA

9. cccDNA conversion • Completion of (+) strand DNA • Exact mechanism is not known • Ability of inhibitor of viral polymerase to block initiation of infection viral polymerase plays a role? • However, also anticipated role of general concepts of cellular DNA repair Activity of viral P protein is not required for cccDNA generation?

10. Recent data (Weizsacker et al., Hepatology) suggested that reverse transcriptase inhibitors can strongly reduce, though not completely block, cccDNA formation - Adefovir: inhibit viral polymerase resulting in a profound suppression of virus production Interferes with plus-strand DNA elongation in incoming particles, thus reducing initial cccDNA formation

11. 2. Removal of P protein at 5’ of (-) DNA • Mechanism of P removal is still obscure • Nucleolytic cleavage of viral DNA close to 5’end by endonuclease OR Hydrolysis of phosphodiester bond between tyrosine residue of polymerase and 5’ phosphoryl group of (-) strand DNA • Whether Deproteinization of RC-DNA occurs before or after its translocation across nuclear membrane is not yet known

12. Cytoplasmic and nuclear lysates prepared • cccDNA only detected in nuclear fraction while DP-rcDNA found in similar amounts in cytoplasm and nuclear fractions • Cytoplasmic DP-rcDNA can be precipitated against HBcAg, suggesting that DNA remains associated with core protein

13. Protein-free RC-DNA (DP-rcDNA): derived from rcDNA upon removal of its covalently attached polymerase protein (Guo et al. 2007, J of virology) • DP-rcDNA exists in both cytoplasm and nucleus, implying that the removal of DNA polymerase from (-) strand occurs in cytoplasm • Majority of cytoplasmic DP-rcDNA present in partially dissembled capsids and minority present within capsids

14. Amplification of cccDNA • Hepadnavirus infected hepatocytes contain up to 50 or more copies of cccDNA • Role for envelope in regulation of cccDNA When sufficient amounts of envelope proteins become available, further cccDNA amplification ceases. (Horwich et al., J of virology) - Accumulation of cccDNA Wild-type virus did not futher accumulate cccDNA after 4 days, while mutant-infected cultures continued to amplify cccDNA

15. 2 possible models to explain suppression of cccDNA synthesis by envelope protein 1. Selection of all capsids for secretory pathway capsidunavilable for cccDNA synthesis 2. Envelope protein may inhibit cellular factors required for cccDNA synthesis : cccDNA synthesis could occur early after infection before sufficient envelope protein is accumulated to inhibit the cellular factors.

16. From cccDNA to pgRNA • Viral RNAs transcribed by cellular RNA polymerase II using cccDNA as template - Includes pregenomic RNA (pgRNA), as well as 3 subgenomic mRNAs. (preS transcript, S transcript, X transcript) - Each gene of HBV has one or more promoters regulating its activity and that these promoters are in turn regulated by one or both of viral enhancer elements, En1 and En2, located upstream of core promoter

17. S2 promoter S2 promoter X promoter Enhancer I Core promoter Enhancer II

18. Regulatory elements of HBV transcription • Precore and pregenomic transcripts • Core promoters direct trascription initiation of both preC and pregenomic mRNA • Core promoter: consists of basal core promoter (BCP) and upstream regulatory sequence • BCP : cis-acting elements that direct precise initiation of both preC and pregenomic RNAs • 2 promoters physically overlap but can function independently : each of pregenomic and preC transcripts produced from distinct promoter sequence

19. Core upstream regulatory sequences (CURS) contains several domains, boxes α, β, γ :These boxes function independently to stimulate BCP activity :However, for functional enhancer II activity, the interaction of both box α and box β is essential • Negative regulatory element (NRE) located further upstream of BCP and can effectively suppress core promoter activity :3 different functional subregions: NRE-α , NRE-β , and NRE-γ :Individually produce a weak suppressive action, but together act synergically to effect an 11-fold inhibitory effect on transcriptional regulation

20. Surface antigen transcripts • Large surface antigen is translated from 2.4kb HBV transcript, while middle and major surface antigens are encoded by 2.1kb mRNA • Large surface antigen transcript is produced under control of S1 promoter • S1 promoter is negatively regulated by sequences located in downstream S2 promoter • 2.1kb middle and major surface antigen transcript is transcribed from S2 promoter

21. S2 promoter contains a CCAAT motif : CCAAT motif not only stimulates production of 2.1kb transcript from S2 promoter but also represses production of 2.4kb transcript • S2 promoter also significantly activated by increased amounts of large surface protein : by indirectly as a consequences of accumulation of large surface protein in endoplasmic reticulum (Mechanism is not clear)

22. Several transcriptional elements identified within S2 promoter (A-G) : Regions A, B, C positively modulate promoter activity : Region D required for maximal activity of promoter : Regions E, F, and G located within 45nt of S2 transcription initiation site : Binding of unidentified transcription factor to region F has negative influence on promoter activity, and this effect is counteracted by factors that bind to region E : Region G though to include transcription initiator sequence

23. HBx transcript • X promoter regulates transcription of small 0.9kb mRNA • Regulatory sequence is located approximately 140nt upstream of transcription initiation site • X promoter contains sites for binding of regulatory protein termed X-promoter binding protein • Tumour suppressor gene product, p53, has been shown to bind to and repress the function of X promoter

24. Pregenomic RNA • pregenomic RNA serves a dual role: • mRNA for core (C) and polymerase (P) synthesis • RNA template for viral genome replication • Early during infection, pgRNA first serves as an mRNA to synthesize C and P proteins • Encapsidation would occur only after C and P protein accumulate above a threshold level

25. Encapsidation of pgRNA • P protein binds to cis-elements on 5’ end of pgRNA, ε(encapsidation signal): Ability of signals to interact with P is closely related to the formation of specific RNA structure • RNA and P protein alter each others conformation • mediates recruitment of core protein dimers and thus leads to packaging of pgRNA-P complex

26. Hydrophobic residues of TP domain of HBV polymerase contribute to viral genome replication (Wang-shickRyu, 2011, FEBS letters) • Mutation in Several conserved hydrophobic residues Analyzed by Southern blot analysis and 3 mutants (W74A, Y147A, Y173A) failed to support genome replication

27. To narrow down specific step of viral genome replication, cytoplasmic total RNA (T), capsid-associated RNA (C) were analyzed by RNase protection analysis - Y173A mutation caused a defect in viral RNA encapsidation, whereas W74A and Y147A mutation caused a defect in viral DNA synthesis

28. 2 processes of translation and encapsidation are competitive, given that they share a common pgRNA (Wang-shickRyu et al., 2007, virology) • pgRNA utilized as mRNA for synthesis of C and P 5’ ε stem-loop disrupted by scanning ribosome As P protein accumulates, it binds to 5’ ε stem-loop structure Suppressing translation Recruitment of core protein dimer by resulting P- complex leads to nucleocapsid assembly

29. Translation suppression by P protein demonstrated by polysome distribution analysis pgRNA associated with polysome fractions was significantly decreased upon P protein expression Decrease in amount of RNA in polysome fractions was accompanied by proportional increase in amount of RNA in nonpolysome fractions

30. Viral RNA remained essentially unaltered upon expression of P protein, Confirming that 5’ εis critically required for translational suppression

31. Reverse transcription of pgRNA • Synthesis of (-)-strand DNA • RNA directed DNA synthesis • HBV uses reverse transcriptase itself as a protein primer for DNA synthesis • DNA synthesis starts by a protein-priming mechanism whereby P protein utilizes a bulged region within as template for a short DNA oligonucleotide whose 5’ end becomes covalently linked to a Tyr resitude in the TP domain of P

32. Polymerase domain • An about 50 amino acid stretch (442~495) in HBV P has no counterpart in DHBV P • The very N terminal TP regions have no significant • homology to each other and are functionally dispensable • The C proximal region is transiently exposed by • chaperone activity and R184 is involved in RNA binding

33. Priming reaction: terminates synthesis of only 4 nucleotides, but it somehow triggers transfer of DNA strand to 3’ end of pgRNA, where 4 nucleotides base pair with complementary sequences

34. DNA primer translocation : DR1* at 3’ end and ε brought into close proximity by closed-loop formation of pgRNA via cellular proteins such as elongation initiation factor, eIF-4G

35. Base pairing between 5’ half of εand cis-Acting sequence φ(Loeb et al., 2007, J of Virology) - 30nt upstream of DR1* called φcan base-pair with left part of the upper εstem - Another element, ω, has been found overlapping with the 3’ end of DR1* - All three elements ε, φ, andωmay interact with each other

36. Completion of (-)-strand DNA • DNA primer extended from DR1* to the 5’ end of pgRNA • RNA simultaneously degraded by RH domain of P protein, except for its capped 5’terminal region including 5’ DR1 • Fate of non-copied 3’ end of pgRNA is not exactly known • P protein continuously attached via its TP domain to 5’ end of (-) DNA strand

37. A model for mechanism of cleavege ( Ganem et al., 1991, EMBO journal) Upon completion of (-) strand DNA synthesis, the active site of RT is positioned at the end of newly completed DNA strand. This in turn positions the active site of Rnase H over 3’ end of DR1, where final cleavages take place resulting in mature (+) strand primer

38. B. (+)-strand synthesis • DNA-directed DNA synthesis : Uses (-)-strand DNA as template RNA frament as a primer - RNA primer translocation: Primer translocates to DR2 and is extended to 5’-end of (-)-DNA

39. Base pairing among cis-acting sequences contributes to template switching (Loeb et al., 2003, PNAS) 11 base pairing 34 base pairing • Sequence identity of donor and acceptor sites contributes to successful template switching • However, other cis-acting sequences make crucial contribution to the (+)-strand template switches • Disrupting base pairing between 3E and M3 and between 5E and M5 inhibits primer translocation and circulization

40. Circulalization • Further elongation requires third template switch • Growing (+)-DNA end is transfered from 5’ r to 3’r on (-)-DNA template and it can further be extended to yield RC-DNA

41. Template switch achieved by small terminal redundancy, r, of the (-) DNA; • 5’r and 3’r are identical over about 8 nt, and consequently both are complementary to the 3’ end of (+) DNA synthesized so far

42. In-situ priming reaction (10% of cases) • Transfer of RNA primer from DR1 to DR2 does not occur Result in double-stranded linear DNA

43. Thank you! ;)