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LF213: Virology > Lecture 11. Hepatits B Virus (HBV) Replication > Flashcards

Lecture 11. Hepatits B Virus (HBV) Replication Flashcards

1
Q

What are hepadnaviruses?

A

Hepatic DNA viruses - all viruses in this class infect hepatocytes

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2
Q

What are examples of orthohepadnaviruses (mammalian)?

A

Human hepatitis B virus
Woodchuck hepatitis virus (used as a model)
Ground squirrel hepatitis virus

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3
Q

What are examples of avihepadnaviruses (avian)?

A

Duck hepatitis B virus (used as a model)
Heron hepatitis B virus

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4
Q

Wy is human hepatitis B difficult to study?

A

Doesn’t grow in a cell culture
Less known about life cycle than some other viruses (most information comes from study on duck and woodchuck hepatitis viruses)

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5
Q

How many people are chronically infected with hepatitis B worldwide?

A

400 million

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6
Q

How many people with hepatitis B will die prematurely?

A

15-25% will die prematurely: hepatocellular carcinoma, cirrhosis

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7
Q

When was hepatitis B first documented and what was it described as?

A

Parenterally transmitted jaundice (serum hepatitis) first documented in 1885

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8
Q

What are hepatitis B virus outbreaks often linked to?

A

Improperly sterilised syringes and needles

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9
Q

What is the timeline from identifying the surface antigen of HBV to vaccine approval?

A

Surface antigen from HBV identified in the serum of patients with serum hepatitis (=hepatitis B) in 1967
HBV particles identified by Dane et al. in 1970
HBV DNA isolated in 1974
Purified surface antigen licensed as a vaccine in 1981

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10
Q

Why was the surface antigen for HBV recognised so early on?

A

There is a lot of it in the serum of infected patients

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11
Q

What shapes can hepatitis B viruses be?

A

Traditional viral particle shape = infectious particles
Small particles and long filaments made up of surface antigen (not infectious)

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12
Q

What are the infectious particles in hepatitis B known as?

A

Dane particles (45nm across)

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13
Q

What are the small particles and long filaments called?

A

Small particle = 22nm HBsAg particle (hepatitis B surface antigen)
Long filaments = pleiomorphic HBsAg filaments

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14
Q

What is the structure of the DNA particle in hepatitis B?

A

Enveloped virus (has membrane) with surface antigen embedded in membrane
Core particle inside the envelope made up of core protein which contains genome
The genome is a relaxed dsDNA
Twice as small as HIV

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15
Q

What is the structure of the HBV genome?

A

Relaxed circular DNA (rcDNA)
3.2 kb partially double stranded
Circularity maintained by overlapping 5’ ends. DR1 and DR2 are direct repeats
(-) strand slightly longer than unit length; (+) strand less than unit length - ends of both strands overlap

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16
Q

What is present on the 5’ end of the -ve sense strand in hepatitis B?

A

Polymerase protein

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17
Q

What is present on the 5’ end of the +ve sense strand?

A

Short RNA oligo and 5’ cap (oligo just means short strand of RNA)

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18
Q

What are the four genes of HBV?

A

P – polymerase
C – core protein
S – surface antigen (3 polypeptides – L, M, S)
X – transactivator of viral transcription

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19
Q

How are the four genes of HBV arranged?

A

Genes are overlapping – the whole genome codes for protein (very compact)

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20
Q

Overview of HBV replication cycle

A
  1. Dane particle attaches to hepatocytes
  2. Virus particle taken in to endosome by endocytosis, fusion of viral and endosomal membranes
  3. Core particle releases into cytoplasm and genome taken to nucleus (uncoating process relatively unknown)
  4. Relaxed circular DNA repaired by DNA repair enzymes in the cells into a covalently closed circular DNA episome, which is used as the template for transcription
  5. Circular DNA episome template generates mRNAs (translated into cytoplasm) and pregenome RNA
  6. Once core proteins and polymerase proteins are encoded, nucleoplasmid with P protein and pregenome forms - core particle forms
  7. Reverse transcriptase occurs, core partical with viral DNA either buds of or renters nucleus
21
Q

What is required for HBV attachment and entry?

A

The large (L) HBsAg

22
Q

What is the cell surface receptor of HBV?

A

The cell surface receptor is NTCP (Na⁺/taurocholate co-transporting polypeptide)

23
Q

How are HBV particles thought to be taken up?

A

HBV particles are thought to be taken up by endocytosis and membrane fusion occurs between viral and cellular membrane. However, the mechanism is unknown

24
Q

How does the core particle release the genomic DNA after travelling to the nucleus?

A

Unknown

25
Q

How is the HBV genome completed?

A

To complete the genome, the P protein is removed from the -ve strand and the capped oligo is removed from the +ve sense strand
DNA polymerases complete the +ve sense strand so that is complementary to the -ve sense strand, becoming covalently closed circular DNA (cccDNA)

26
Q

What is covalently closed circular DNA (cccDNA) the template for?

A

Transcription by host RNA Polymerase II
HBV DNA contains a transcription enhancer that is only active in liver cells.

27
Q

What is HBV X and how does it function?

A

A transcriptional activator;
Doesn’t bind DNA itself
Activates transcription factors
Interacts with cell signalling pathways

28
Q

What happens in HBV transcription?

A

mRNAs are capped and polyadenylated, but not spliced (no introns, no splice signals)
Nuclear export of mRNAs to the cytoplasm depends on a post-transcriptional regulatory element (PRE) that uses the CRM1/RanGTP pathway (like Rev/RRE). But no viral protein is required (no protein equivalent to Rev to mediate the reaction)

29
Q

What are the four classes of mRNAs that are produced in HBV transcription?

A

3.5 kb – pregenomic RNA (template for making genome), encodes P and C proteins
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

30
Q

What do all four classes of mRNAs produced by HBV transcription have in common?

A

All end at a common polyadenylation (polyA) site despite having different transcription start sites

31
Q

How does the 3.5 kb mRNA encodes C and P?

A

Translated by leaky scanning, some ribosomes will start at start codon for P located after the start codon for C proteins

32
Q

How many mRNAs are there that are 2.1 kb in length during HBV transcription?

A

Two mRNAs that have slightly different start sites
One enables translation for medium surface antigen (M) and the other small surface antigen (S)

33
Q

What is the process of encapsidation of pregenomic RNA into the core particle?

A
  1. Inactive P protein with reverse transcriptase (RT) and terminal protein (TP) domains
  2. Becomes activated by the binding of cellular chaperone and virus X proteins inducing conformational change in P protein
  3. Conformational change allows P protein to bind to ε element of pregenomic RNA, the P protein being bound to RNA is the signal for assembly of the core particle
  4. Core proteins start assembling around the P:RNA complex, forming capsid like structure
34
Q

What is the difference between HBV and HIV reverse transcription?

A

HBV reverse transcription takes place in a newly formed core particle unlike HIV that takes place in a partially disassembled capsid

35
Q

What is the primer for reverse transcriptase in HBV?

A

The terminal domain of the P protein (tyrosine 96)
How the P protein is covalently linked to the -ve strand of the DNA

36
Q

Because of the direct repeats on the +ve and -ve strands (DR1 and 2), what can the primer do during HBV replication?

A

Primer moves to template 3’ end of -ve DNA, allowing DNA synthesis all around the pregenome RNA

37
Q

What happens when the -ve strand of DNA is completed?

A

RNA template is degraded; 5’ capped oligo spared from RNase H activity as acts as a primer for +ve DNA

38
Q

What happens after the RNA template is degraded in HBV?

A

Capped oligo pairs/jumps to DR2, imitating +ve DNA that is complimentary to the -ve strand
This happens until the +DNA pairs with DR 1 at the 3’ end of -DNA, allowing extension to continue

39
Q

Why does the +ve DNA end only 2/3 round the circular DNA during HBV reverse transcription?

A

Runs out of deoxyribonuclease

40
Q

What are the similarities between HIV and HBV reverse transcriptase?

A

Both enzymes have a reverse transcriptase (RNA-dependent DNA polymerase) domain and an RNase H domain

41
Q

What are the differences between HIV and HBV reverse transcriptase?

A

Utilise different priemrs
Terminal protein of HBV P protein is the primer for (-) strand DNA synthesis – first nucleotide is added to tyrosine 96. HIV RT uses tRNAlys as the primer for (-) strand DNA synthesis

42
Q

What is the difference between the viral genome in HIV and HBV reverse transcription?

A

HIV viral genome = RNA (pseudodiploid)
HBV viral genome = DNA (incomplete duplex)

43
Q

What is the difference between the other role of the template RNA in HIV and HBV reverse transcription?

A

HIV template RNA also = genome RNA, mRNA (gag,pol)
HBV template RNA also = pregenome RNA, mRNA (C and P proteins)

44
Q

What is the difference between the functions in HIV and HBV reverse transcription?

A

HIV reverse transcriptase functions = DNA polymerase, RNase H, helicase (strand displacement)
HBV reverse transcriptase functions = DNA polymerase, RNase H, protein priming, template RNA encapsidation

45
Q

What is the difference between the reverse transcribing nucleoprotein complexes in HIV and HBV reverse transcription?

A

HIV reverse transcribing nucleoprotein complexes = subviral “core” particles, deposited in the cytoplasm on viral entry
HBV reverse transcribing nucleoprotein complexes = Nascent subviral “cores” cytoplasmic intermediates in viral assembly

46
Q

What is the difference between the final products in HIV and HBV reverse transcription?

A

HIV final product = double-stranded linear DNA
HBV final product = circular viral DNA or covalently closed episomal DNA

47
Q

What is the difference between the DNA maintained in the nucleus in HIV and HBV reverse transcription?

A

HIV DNA maintained in the nucleus = integrated into host genome, proviral DNA
HBV DNA maintained in the nucleus = nonintegrated episome in host nucleus

48
Q

How are HBV viruses assembled?

A

The concentration of L (large) surface antigen determines the fate of core particles
L, M, S surface antigens accumulate in ER-Golgi membranes. C (core) protein interacts with L
Low concentration of L leads to the core particle/viral DNA entering the nucleus, where it is converted to cccDNA
There can be up to 10-20 copies of cccDNA in one cell nucleus. Each can be a template for transcription
High concentration of L leads to the core particle budding into the ER. Virions exit the cell by exocytosis (also can get empty particles containing small surface antigen)
Empty HBsAg particles (containing mostly S) also bud into the ER and undergo exocytosis

LF213: Virology (14 decks)

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