Hepnadaviruses

Question 1

What is an example of a hepnadavirus

Answer 1

Hepatitis B

Question 2

What is the general structure of Hepatitis B?

Answer 2

Partially ss/dsDNA, has an RNA segment
Undergoes retrotranscription but usually doesn’t integrate (doesn’t encode an integrase but does occasionally happen)
Can cause cancer

Question 3

What are the different hepatitis viruses?

Answer 3

Hep A: picorna virus, food/blood bourne, good vaccine, bad drugs
Hep B: Good vaccine but prevalent, major cause of liver cancer
Hep C: good drugs, bad vaccine
Hep D: only replicate in cells infected with hep B, makes disease worse
Hep E: from contaminated pork, only serious during pregnancy

Question 4

How is HBV spread?

Answer 4

Via bodily fluids e.g. in birth, sex, sharing needles, sharing razors/toothbrushes, contact with blood or sores

Question 5

What are the symptoms of HBV?

Answer 5

70% are asymptomatic
30% develop acute infection with symptoms such as abdominal pain, dark urine, fever, joint pain, nausea and vomiting, jaundice
Symptoms appear over a month after exposure
Acute infection lasts 1-3 months, chronic infection can develop in 1% of cases and leads to liver cancer

Question 6

How can we study HBV?

Answer 6

Doesn’t grow well in cell culture, so have to use animal models such as woodchuck, squirrel, pekin duck and heron. Not perfect as each HBV has a narrow host range

Question 7

What is the HBV virion structure?

Answer 7

Virion referred to as a Dane particle. Is enveloped and contains HBV surface antigen (HBsAg), Core (HBcAg) and a polymerase linked to the genome. Is an icosahedral virus with 240 copies of HBcAg

Question 8

What types of particles does HBV produce?

Answer 8

1 - the Dane particle with equimolar amounts of all 6 HBsAg (L, M and S either glycosylated or not)
2- filamentous particles containing gp27/p24 (S) and some gp36/p33 (M)
2 - sphere containing only gp27/p24 (S)
The filamentous particles and the sphere are immunological decoys

Question 9

Describe the HBV genome

Answer 9

Very small - 3.2kb of partially ds DNA
The negative sense strand is complete but contains a nick and is linked to the polymerase at the 5’ end
The positive sense strand is incomplete and has a short capped RNA oligonucleotide attached at the 5’ end

Question 10

What does the HBV genome encode?

Answer 10

4 overlapping ORFs. S - surface antigen, P - polymerase, X - multifunctional and C - Core

Question 11

How does HBV enter the cell?

Answer 11

Binds to sodium taurocholate cotransporting polypeptide (NTCP). The genome is then delivered to the nucleus in a poorly characterised manner. In the nucleus, the relaxed circular (rc) DNA is converted to covalently closed circular (ccc) DNA by host enzymes in a repair process. This is very stable and is maintained and replicated episomally.

Question 12

Describe HBV transcription

Answer 12

Once the DNA has been converted to the cccDNA form, transcription begins.
RNA pol II produces capped and polyadenylated transcripts
At least 5 transcripts are made: 3 subgeneric RNAs, 1 pregenomic RNA (pgRNA) and 1 PreC RNA.
All are translated, the pgRNA is a template for genome replication
Likely to be a 6th alternative spliced RNA

Question 13

How is the pgRNA transcribed?

Answer 13

The promoter for the pre genomic RNA starts upstream of the poly A sequence and bypasses it the first time. It terminates the second time. It is the template for reverse transcription. Bypasses as it is very close to the promoter. Suggested that the RNAPII is ‘super active’ and ignores the first few hundred bases. The 5’ end is the same as the 3’ end.

Question 14

Describe the HBV polymerase

Answer 14

Has 3 activities: priming, reverse transcriptase and RNaseH. Is a multi domain protein

Question 15

How does priming work in HBV, using the pgRNA template?

Answer 15

The reverse transcriptase binds epsilon (region on the pgRNA), and 4 DNA nucleotides are added to the TP domain of the reverse transcriptase. Template is a loop at the top of the epsilon stem (see diagram). the pgRNA is encapsidated here, before reverse transcription is complete.

Question 16

Describe the first primer shift in HBV

Answer 16

The reverse transcriptase moves to base pair with the DR1 region of the pgRNA, facilitated by RNA circularisation (due to eIF4F and PABP). This triggers synthesis of the negative strand of DNA, with the polymerase RNase H activity degrading the RNA as the DNA is made .A small RNA primer is left at the 3’ end where it hasn’t been synthesised (DR1 is placed slightly inside the 3’ end)

Question 17

Describe the second primer shift in HBV

Answer 17

The short capped primer (left from the RNase H activity) moves to base pair with a region downstream of DR2 (at the 5’ end). This is used as a primer for DNA synthesis -only a short strand - followed by circularisation due to sequence complementarity. Positive strand DNA synthesis continues but doesn’t go to completion - thought to be due to dNTP concentration is limited in the virion capsid

Question 18

Describe HBV packaging

Answer 18

P protein to epsilon (cis element in pgRNA) binding leads to recruitment of the core dimer and packaging of the P-pgRNA complex. After DNA synthesis, the core buds into the ER and is released by exocytosis, potentially involving components of the ESCRT pathway

Question 19

What is the importance of type 1 interferons in HBV infection?

Answer 19

It is poorly produced - the virus suppresses it.

Question 20

What is the importance ofT cells in HBV infection?

Answer 20

Depletion of CD4 and CD8 T cells in chimpanzees showed that CD8 T cells are the main effector cells, but CD4 T cells are required early on to induce an efficient CD8 T cell response. Weak CD8 T cell responses are seen in chronic HBV patients

Question 21

How can HBV establish a persistent infection after intrauterine infection/antigen transfer?

Answer 21

Clonal deletion of antigen specific T cells

Remaining cells tolerised by liver dos conditioned by maternal HbeAg exposure to express PD-L1

Question 22

How can HBV establish a persistent infection after intrapartum/neonatal infection?

Answer 22

In most individuals the immature immune response is unable to control virus replication until virus has spread throughout the liver. Many virus-specific T cells are then exhausted

Question 23

What are the consequences of HBV persistence?

Answer 23

Immune complex disease
Chronic hepatitis (CD8 T cells fail to control viral replication, other immune cells are recruited  such as lymphocytes, macrophages, neutrophils that cause liver damage by antigen non-specific mechanisms)
Hepatocellular carcinoma, driven randomly or by integration of viral genome.

Question 24

How can HBV establish persistent infection in adults?

Answer 24

If the T cell response isn’t strong enough, results in T cell exhaustion. Only 10% of cases

Question 25

How can HBV be studied in mice?

Answer 25

HBV doesn’t infect mice, so instead make transgenic mice with HBV genes - drives expression of virus in liver (and a little in kidneys). Virus is shed in urine. Mice are tolerant as they see the virus as self.

Question 26

What is the importance of CD8 T cells in HBV infections?

Answer 26

Transgenic HBV mice with CD8 T cells from an immunised mice - show that CD8 T cells do perforin dependent lysis of hepatocytes, but perforin isn’t required (can control infection without liver damage). Infection is controlled by IFNgamma and TNFalpha

Question 27

How can HBV infections be controlled?

Answer 27

If HBV transgenic mice are infected with LCMV get good control of HBV as well - shows that LCMV response is useful for HBV. Similar thing in humans with Hep B and Hep C infection - antiviral cytokines and type 1 interferons are very good
Can also use HIV reverse transcriptase inhibitors against HBV
In woodchuck infection, can vaccinate against HBV along with blocking PDL-1 to prevent T cell exhaustion or using IL-12 as an adjuvant (to rescue exhausted T cells) can enhance T cell control of HBV. If given with antiviral agents can also get viral clearance