Flavivirus Replication Cycle Flashcards
1
Q
What is the genome of a DENV virion?
A
Positive sense ssRNA genome
2
Q
How big is the DENV genome?
A
Around 10-11 kb
3
Q
What is the membrane of a DENV virion derived from?
A
Derived from the host cell
4
Q
What is the function of the envelope protein?
A
Mediates cell fusion/entry
5
Q
How is the envelope protein organised?
A
- Organised into 3 dimers held together
- Each plate is made up of 6 copies of the envelope protein
- The dimer has 3 domains - domain I, domain II and domain III
6
Q
What is the function of pre-membrane protein?
A
- Assists E protein folding
- Prevents premature fusion of immature particles
7
Q
Describe the Flaviviridae life cycle
A
- Virus binds to host receptor
- Receptor-mediated endocytosis
- Endosome is acidified releasing a nucleocapsid - nucleic acid surrounded by the capsid and enveloped in the E protein
- Virus goes through translation to produce a polyprotein which all other viral proteins are derived from - happens on membranes derived from the ER
- Genome is replicated
- Assembly of viral particles - genome is encapsulated with capsid protein
- Buds through the ER
- Released through the secretory pathway
- Flaviviruses don’t burst the cell so can have consistent release - allows for persistent infections
8
Q
Describe the viral rearrangement during DENV fusion
A
- Start with a mature viral particle
- During fusion there will be a major rearrangement of the viral particle - more spiky and more open
- Rearrangement allows the virus to fuse with the cell membrane and eject its nucleic acids into the cell
- Change from 3 dimer formation to 2 trimers
9
Q
Describe DENV membrane fusion
A
- Fusion peptide is buried inside the E protein dimer on the virion surface - mediates fusion with the membrane to allow the virus to enter the cell
- After receptor-mediated endocytosis, acidification of the endosome causes E protein to swing open towards the host cell membrane
- Insertion of E protein into the host cell membrane and timerisation of E proteins on viral membrane
- Further conformational change in the E protein pulls host and viral membranes together
- Hemifusion = just one of the lipid layers of each membrane has fused
- Full fusion = both lipid layers have fused - creates a pore allowing the contents of the viral particle to enter the cell
10
Q
Describe the Flaviviridae genome structure
A
- Positive sense ssRNA genome which is directly translated upon entry into the cell - can directly use host machinery
- Hepaciviruses don’t have a cap - mRNA does
- Flaviviruses and hepaciviruses don’t have a poly(A)tail - mRNA does
11
Q
How is the DENV polyprotein processed?
A
- Polyprotein is processed into 10 protein by proteolytic cleavage done by proteases
- 3 structural proteins - part of the virion - capsid, prM and E
- 7 non-structural - present in the virally infected cell but not present in the viral particle
- Viruses encode their own protease - NS3 in dengue in combination with cofactor NS2B
- The protease cleaves the polyprotein on the cytoplasmic side of the ER membrane
- On the ER lumen side the polyprotein is cleaved by signalases and furin
12
Q
How is the positive sense ssRNA genome replicated?
A
- Produces a negative sense RNA genome first - intermediate made using positive sense ssRNA as a template
- The negative sense ssRNA can be used as a template to make positive sense ssRNA
- Translation occurs 5’ to 3’
- Genome replication occurs 3’ to 5’
- So translation and genome replication are mutually exclusive - can’t use the same RNA molecule as the translation and replication machinery would collide
13
Q
What are the viral factors involved in genome replication?
A
- Cells don’t encode an RNA-dependent RNA polymerase - as cells have DNA dependent RNA polymerases
- NS5 = methyltransferase - involved in mRNA capping
- NS3 = multifunctional - helicase – helps unwind the RNA - NTPase - 5’ triphosphatase – involved in mRNA capping
- NS1 = cofactor
- These proteins form the replication complex
14
Q
How does the switch from positive to negative strand synthesis occur?
A
- ER membrane reorganisation regulates switch between positive and negative strand synthesis
- Early in infection, replication complexes associated with ER membranes - favours negative strand synthesis
- Late in infection, non-structural proteins reorganise ER membranes to concentrate negative ssRNA and replication complexes - concentration into almost vesicles favours positive strand synthesis
15
Q
Describe viral assembly
A
- Capsid/core protein packages viral RNA and buds into the ER lumen
- Capsid protein forms on the cytoplasmic side of the ER membrane
- Have envelope proteins on the inside of the ER
- As positive RNA buds into the ER get the membrane rom the cell forming around the viral RNA genome
- Immature viral particles are contained within vesicles and released via secretory pathway - goes through the golgi and transported in vesicles
- Therefore, Flaviviridae aren’t lytic viruses - not released by lysis of the cell
