L14/15 HepC (2) Flashcards
(39 cards)
How is the infectious molecular clone of HCV made?
pHCV plasmid with T7 promoter
dsDNA made by RT-PCR
T7 promoter recognised by RNA polymerase from bacteriophage T7
generate RNA molecules almost same as present in virus particle
inject in chimp
levels of RNA in serum you would see in infected serum
Why are infectious clones of HCV useful?
allow studies on individual viral components
shows all enzymatic activities are needed for replication
identifies critical RNA sequences in UTRs
Does RNA derived from infectious clones replicate in cultured cells?
NO
How is a sub-genomic replicon of HCV constructed?
Removal of code for STRUCTURAL proteins
Clone in gene NPT
IRES
What is NPT?
Neomycin phosphotransferase
gives resistance to antibiotics neomycin & kanamycin
What is G418 selection?
artificial analogue of neomycin - resistance selection
What is the control for G418 selection?
mutate active site by reverse genetics
mutate GDD to GND in NS5B polymerase to kill it
How are replicon-harbouring cells harvested?
G418 selection
What happens in a replicon harbouring cell?
IRES drives translation
NPT phosphorylates G418 (usually inhibits translation)
What does the subgenomic replicon tell us?
5 NS proteins are needed for RNA replication
NS3-5B forms multi-protein replication complex
Replicates RNA - 5’ 3’ UTRs
Replication occurs in cytoplasmic membrane bound compartment
Problems with subgenomic HCV replicons?
Only work in Huh7 cells
ONLY study the replication and translation part of the life cycle
What was the major breakthrough in HCV studies?
genotype 2a derived from patient with Japanese Fulminant Hepatitis (JFH-1)
Sub-genomic replicons of JFH-1 replicate WITHOUT culture adaptation - did NOT need to pick up mutations
full length transfection leads to infectious virus particles
Proteins involved in HCV ENTRY?
GAG LDL-R SRB1 CD81 Claudin Occludin
What side of the ER membrane is the HCV core protein situated on?
cytoplasmic side
What side of the ER membrane are the HCV E1/E2 proteins situated on?
ER lumen side
What is NS2 protein?
Cleaves itself from NS2-3 precursor
cytoplasmic domain dimerises
What does NS2 protein do?
virus assembly & release
ALSO other functions
NOT involved in replication
What is NS3/4A protein?
Bi-functional enzyme
PROTEASE and HELICASE activity
targeted to membrane due to NS4A
What 2 things does NS3 do?
mediates cleavage between all other NS proteins
block antiviral innate immune response
What does NS4A do?
promotes protease activity of NS3
How are the structural proteins cleaved?
Cellular proteases
How are the non-structural proteins cleaved?
NS3 cleavage and NS2/3 autocleavage
What does NS3/4A block?
IFN-b expression:
RIG-I and TLR3 detect dsRNA
TLR3 usually activates TRIF pathway
RIG-I usually activates MAVS pathway
HCV cleaves TRIF and MAVS = blocks IRF-3 and stops IFN-b production
What 2 cellular pathways does HCV cleave?
TRIF
MAVS
