Flavivirus Replication Cycle

Question 1

What is the genome of a DENV virion?

Answer 1

Positive sense ssRNA genome

Question 2

How big is the DENV genome?

Answer 2

Around 10-11 kb

Question 3

What is the membrane of a DENV virion derived from?

Answer 3

Derived from the host cell

Question 4

What is the function of the envelope protein?

Answer 4

Mediates cell fusion/entry

Question 5

How is the envelope protein organised?

Answer 5

• 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

Question 6

What is the function of pre-membrane protein?

Answer 6

• Assists E protein folding

- Prevents premature fusion of immature particles

Question 7

Describe the Flaviviridae life cycle

Answer 7

• 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

Question 8

Describe the viral rearrangement during DENV fusion

Answer 8

• 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

Question 9

Describe DENV membrane fusion

Answer 9

• 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

Question 10

Describe the Flaviviridae genome structure

Answer 10

• 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

Question 11

How is the DENV polyprotein processed?

Answer 11

• 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

Question 12

How is the positive sense ssRNA genome replicated?

Answer 12

• 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

Question 13

What are the viral factors involved in genome replication?

Answer 13

• 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

Question 14

How does the switch from positive to negative strand synthesis occur?

Answer 14

• 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

Question 15

Describe viral assembly

Answer 15

• 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

Question 16

How do DENV particles mature?

Answer 16

• In immature particles, pre-M protein covers the fusion loop on E, preventing premature fusion before viruses are released - spiky appearance - prM is on top of the fusion peptide
• Furin cleavage of pre-M into M causes conformational changes in E and matures the virus particle as it’s released from the cell