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
