Hepatitis B Virus Replication (11) Flashcards
What class of virus is human hepatitis B virus (HBV) in?
Class 7 viruses: Hepadnaviruses
→ infect hepatocytes of mammals/birds
Orthohepadnaviruses: human hepatitis B virus, woodchuck hepatitis virus*, ground squirrel hepatitis virus
Avihepadnaviruses: duck hepatitis B virus, heron hepatitis B virus
HBV is difficult to study, doesn’t grow in cell culture → less is known about the life cycle
*useful models - most information comes from studying these
What is the burden of hepatitis B virus?
400 million people chronically infected worldwide
→ 15-25% will die prematurely: hepatocellular carcinoma, cirrhosis
→ burden concentrated in subsaharan Africa
→ also highly found in Alaska, northern Canada and throughout Asia
When was hepatitis B virus (HBV) discovered?
1885 → parentally transmitted jaundice (serum hepatitis) first documented
→ outbreaks linked to improperly sterilised syringes and needles
1967 → surface antigen from HBV identified in the serum of patients with serum hepatitis
1970 → HBV particles identified by DANE et al.
1974 → HBV DNA isolated
1981 → purified surface antigen licensed as a vaccine (very successful)
→ there is a lot of surface antigen in serum of infected people - allowing it to be isolated early on
What are the types of hepatitis B virus particle?
45nm Dane particle → infectious virion, enveloped virus with surface antigen embedded,
22nm HBsAg particle → small
Pleiomorphic HBsAg long filaments
→ HBsAg = hepatitis B surface antigen
→ not infectious, just consist of HBsAg - easily identifiable, good for vaccine
What is the hepatitis B virus (HBV) genome?
Relaxed circular DNA (rcDNA)
→ 3.2kb partially double stranded DNA
→ -ve strand slightly longer than unit length, +ve strand slightly shorter (doesn’t stretch round the whole genome circle)
→ circle maintained by overlapping 5’ ends
→ contains DR1 and DR2 - direct repeats, identical sequences
Only consists of 4 genes - overlapping → entire genome codes for protein - very compact
P → polymerase
C → core protein
S → surface antigen (2 polypeptides - L, M, S)
X → transactivator of viral transcription
What are the key events in the hepatitis B virus (HBV) life cycle?
→ attachment and entry
→ penetration of the nucleus
→ genome conversion to covalently closed circle
→ transcription producing mRNA and pregenome
→ caspid assembly and pregenome reverse transcription
→ virus envelopment by budding into ER, then exocytosis
→ OR re-entry into nucleus, amplifying DNA copy number
What is involved in hepatitis B virus (HBV) entry and uncoating?
Hepadnaviruses have a very narrow host range - only infect hepatocytes
→ the large (L) HBsAg is required for attachment and entry (M and S don’t interact with receptor)
→ the cell surface receptor is NTCP (Na+/taurocholate co-transporting polypeptide)
HBV particles are thought to be taken up by endocytosis
→ membrane fusion occurs between viral and cellular membrane - mechanism unknown
Core particle travels to he nucleus and releases the genomic DNA
→ mechanism of uncoating is unknown
What is involved in hepatitis B virus (HBV) genome completion?
Genome completion = converting relaxed circular DNA to covalently closed circular cccDNA
Incoming particle genome has capped oligo on 5’ +ve sense strand and polymerase on 5’ -ve sense strand
→ P protein and cap oligo removed, DNA polymerases complete the short +ve strand
→ carried out by cellular enzymes in the nucleus
What is involved in hepatitis B virus (HBV) transcription?
cccDNA is a template for transcription by host RNA Polymerase II
→ HBV DNA contains a transcription enhancer that is only active in liver cells
HBV X protein is a transcriptional activator
→ not a traditional transcription factor - doesn’t bind DNA itself, activates cellular transcription factors, interacts with cell signalling pathways
The mRNAs produces are capped and polyadenylated - but not spliced
→ can’t use normal cellular nuclear export pathways - depends on post-transcriptional regulatory element (PRE) that uses the CRM1/RanGTP pathway (like Rev/RRE) but not viral protein is required
What does transcription of hepatitis B virus (HBV) produce?
4 classes of mRNAs
3.5 kb → pregenomic RNA, mRNA encoding core protein and polymerase protein (C & P)
2.4 kb → encodes large (L) surface antigen
2.1 kb → encodes medium (M) and small (S) surface antigens
0.7 kb → encodes X protein
→ have different transcriptional start sites - different promoter elements within the DNA, but have common poly A site
How are the different mRNAs of hepatitis B virus (HBV) translated?
Viral RNAs can encode more than one protein - make the most of their coding capacity
3.5 kb → leaking scanning
→ most ribosomes will initiate translation at first AUG producing core protein
→ some will bypass first AUG keep scanning to downstream AUG - initiate translation of polymerase
→ evolved to produce appropriate ratios for the virus (needs more core)
2.4 kb → produces 3 domains of L surface antigen
2.1 kb → 2 mRNAs ~2.1 kb with slightly different start sites produce M and S surface antigens
What is involved in the encapsidation of pregenomic RNA into core particle of hepatitis B virus (HBV)?
Once C and P proteins are made - start to get encapsidation of pregenomic RNA
P proteins has 2 domains: RT - reverse transcriptase, TP - terminal protein
→ becomes activated by binding of some cellular chaperone proteins and virus X protein - induces conformational changes allowing it to bind to pregenomic RNA at a particular site called ε
→ P protein bound to RNA at ε - signal for core particle assembly - capsid structure around RNA
→ reverse transcription can occur in the newly made caspid
What is involved in hepatitis B virus (HBV) genome production (reverse transcription)?
Pregenome RNA made in nucleus moves into cytoplasm
P protein bound to ε of pregenome RNA initiates -ve DNA strand synthesis inside core particle
→ can’t start without a primer, terminal protein domain of P protein is the primer (tyrosine 96aa - where the first nucleotide is added)
Initial DNA made is short - template jump from direct repeat sequences, to 3’ end, -ve DNA extended
→ -ve strand of DNA complementary to the pregenome RNA
Reverse transcriptase has RNaseH acitivty
→ degrades RNA in duplex with DNA - pregenome RNA degraded all the way round (other than 5’ capped oligo - acts as primer for +ve sense DNA to be made)
5’ capped oligo can initial a template jump to other end of -ve strand
→ synthesise DNA complementary to the -ve strand
→ gets to DR1 switches template and starts to copy - extension of the +ve sense strand
Continues until +ve strand ~ 2/3 complete
→ running out of deoxynucleotides to add
How do the reverse transcriptase of HBV and HIV compare?
There is some homology between HIV and HBV enzymes
→ both contain reverse transcriptase domain (RNA-dependant DNA polymerase) and RNasH domain
→ some aa sequences common between the proteins - share some key catalytic aa
difference: HPV has terminal protein domain and a spacer
What are the two pathways a hepatitis B virus (HBV) core particles with viral DNA can take?
Core particles thats reverse transcribed genome DNA can go:
1. back into nucleus - repaired to covalently closed circle, amplifying DNA copy number
2. can bud into ER Golgi, get enveloped - move out of cell via secretory pathway
Path taken determined by conc. of large surface antigen
→ early on when low [L] made - will return to nucleus
→ later on in higher [L] - it will leave cell
What happens to empty HBsAg particles of HBV?
Emty HBsAg particles (containly mostly S) bud into the ER and undergo exocytosis
→ secretion of empty particles containing small surface antigen
→ probably aids virus in mopping up of host immune response
What is the key difference between retrovirus (HIV) and hepadnavirus (HPV) life cycles?
They are similar but there are key differences in timings are where RT happens in life cycle
Retrovirus: (+)RNA enters → reverse transcription - viral enzyme makes dsDNA → transcription - host RNA pol II makes RNA
Hepadnavirus: dsDNA enters → transcription - host RNA pol ii makes (+)RNA → reverse transcription - viral enzyme makes dsDNA
