Hepadnaviridae

Question 1

What is the structure of the virus particle?

Answer 1

• The Hepatitis B virus particle has an enveloped layer with surface antigen (HBsAg).
• It has a core protein that makes up the capsid.
• Internally, it has the genome which is dsDNA that is assembled with reverse transcriptase.
• It is 42 nm.
• There are also incomplete forms of the virus that serve as decoys to divert antibody immunity and they are non-infectious particles.
• They are 15-25 nm or 20 x 20-200 nm.

Question 2

What is the genome structure?

Answer 2

• The genome is dsDNA and it is 3.2 kb in size.
• It is a relaxed circle that is incomplete and has viral polymerase bound -> this viral polymerase is RNA dependent DNA polymerase but in some cases can be DNA dependent DNA polymerase.
• There are two short direct repeat sequences (DR1 and DR2) of 12bp that have complementarity and a short capped RNA primer of 18 nucleotides.
• It forms an incomplete circle.

Question 3

How many mRNAs can be produced from the genome?

Answer 3

The dsDNA genome of HBV can transcribe four different sized mRNAs using four different dsDNA promoter/enhancer transcription start sites. Each of these terminate at the same place with a poly(A) tail.
There is ORF C, ORFX, ORFP and ORFS.

Question 4

What proteins are encoded for by the pregenomic RNA?

Answer 4

Pre-genomic RNA goes more than one lap around the circular genome and is 3.4 kb in size. It is capped and polyadenylated so it can serve as mRNA and it encodes several different proteins. It encodes for the pre-core protein and the core protein is produced to make the capsid structure. From a different reading frame, it can encode for the polymerase enzyme. It produces all of the structural proteins within the core of the virus.

Question 5

What do the other mRNA’s code for?

Answer 5

The shorter mRNA’s produced can code for a series of other proteins such as the S proteins that become the surface antigens present on the envelope.
There is also another RNA that is 0.7 kb that codes for the X antigen.

Question 6

How does HBV attach to cells?

Answer 6

The hepatitis B virus attaches to a specific receptor on hepatocytes known as NTCP and releases the core.

Question 7

How does HBV enter the cells?

Answer 7

The virion is endocytosed and then the nucleocapsid is released from the endosome by fusion of the virion and endosome membranes. The nucleocapsid is transported to the nucleus on a microtubule.

Question 8

What occurs to the HBV genome once it is inside the nucleus?

Answer 8

Once the virus genome is in the nucleus it is converted into a circular DNA molecule as nucleotides are available and a cccDNA forms (covalently closed circular DNA). The cccDNA is the template for transcription and it is carried out by RNA polymerase II. The genome has four promoters upstream of the pre-S1, pre-S2, X and pre-C regions and mRNAs are produced from these. The pregenomic RNA is the most important produced and this is longer than the genome itself. During synthesis, part of the genome is transcribed twice and these RNAs have direct terminal repeats. The RNA polymerase must therefore ignore the polyadenylation signal the first time.

Question 9

What happens with translation?

Answer 9

The pregenomic RNA is then translated forming the core proteins and polymerase. The C protein forms dimers, which are assembled into nucleocapsids and the polymerase (P protein) and pregenome are within these.

Question 10

What happens with reverse transcription?

Answer 10

DNA synthesis takes place in the capsid and the pregenome RNA is the template and DNA is synthesised by the reverse transcriptase domain of P. The terminal protein domain of P acts as the primer for the initiation of (-) strand DNA synthesis. The pregenomic RNA also has terminal redundancy (or repeats - DR1 and DR2).

• Initiation of (-) DNA synthesis near the 5’ end of the pregenome RNA.
• Transfer of DNA to the same complementary sequence in DR1 near the 3’ end of the pregenome.
• Continuation of (-) DNA synthesis and degradation of the pregenome by RNase H (polymerase activity).
• Completion of (-) DNA synthesis. All of the RNA has been degraded, except for the a short sequence at the 5’ end.
• Transfer of RNA to the same complementary sequence near the 5’ end of the (-) DNA.
• Synthesis of (+) DNA on the circularised (-) DNA template.
• The elongation of the strand happens 5-10% of the time and this becomes cccDNA. Or you can get re-annealing of the primer to the DR2 (occurs 90-95%). The growing DNA strand will read through DR1 and then you get a second switch in the template and it is able to elongate against the negative strand. This is the dsDNA genome encapsulated within new particles.