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MICR2222 - Medical Virology > L14/15 HepC (2) > Flashcards

L14/15 HepC (2) Flashcards

1
Q

How is the infectious molecular clone of HCV made?

A

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

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2
Q

Why are infectious clones of HCV useful?

A

allow studies on individual viral components

shows all enzymatic activities are needed for replication

identifies critical RNA sequences in UTRs

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3
Q

Does RNA derived from infectious clones replicate in cultured cells?

A

NO

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4
Q

How is a sub-genomic replicon of HCV constructed?

A

Removal of code for STRUCTURAL proteins

Clone in gene NPT

IRES

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5
Q

What is NPT?

A

Neomycin phosphotransferase

gives resistance to antibiotics neomycin & kanamycin

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6
Q

What is G418 selection?

A

artificial analogue of neomycin - resistance selection

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7
Q

What is the control for G418 selection?

A

mutate active site by reverse genetics

mutate GDD to GND in NS5B polymerase to kill it

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8
Q

How are replicon-harbouring cells harvested?

A

G418 selection

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9
Q

What happens in a replicon harbouring cell?

A

IRES drives translation

NPT phosphorylates G418 (usually inhibits translation)

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10
Q

What does the subgenomic replicon tell us?

A

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

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11
Q

Problems with subgenomic HCV replicons?

A

Only work in Huh7 cells

ONLY study the replication and translation part of the life cycle

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12
Q

What was the major breakthrough in HCV studies?

A

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

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13
Q

Proteins involved in HCV ENTRY?

A 
GAG
LDL-R
SRB1
CD81
Claudin
Occludin
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14
Q

What side of the ER membrane is the HCV core protein situated on?

A

cytoplasmic side

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15
Q

What side of the ER membrane are the HCV E1/E2 proteins situated on?

A

ER lumen side

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16
Q

What is NS2 protein?

A

Cleaves itself from NS2-3 precursor

cytoplasmic domain dimerises

17
Q

What does NS2 protein do?

A

virus assembly & release

ALSO other functions

NOT involved in replication

18
Q

What is NS3/4A protein?

A

Bi-functional enzyme

PROTEASE and HELICASE activity

targeted to membrane due to NS4A

19
Q

What 2 things does NS3 do?

A

mediates cleavage between all other NS proteins

block antiviral innate immune response

20
Q

What does NS4A do?

A

promotes protease activity of NS3

21
Q

How are the structural proteins cleaved?

A

Cellular proteases

22
Q

How are the non-structural proteins cleaved?

A

NS3 cleavage and NS2/3 autocleavage

23
Q

What does NS3/4A block?

A

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

24
Q

What 2 cellular pathways does HCV cleave?

25
Q

WHat does TLR3 detection trigger?

26
Q

What does RIG-I detection trigger?

27
Q

What is NS4B?

A

highly hydrophobic membrane spanning protein

28
Q

Role of NS4B?

A

Changes structure of membrane within cell

induces membrane rearrangement - membranous web

29
Q

What is NS5A?

A

3 domains bound by linker region

D1 - Zn molecule, disulphide bond

amphipathic helix N terminus - sits on membrane due to hydrophobic region

30
Q

Role of NS5A?

A

RNA replication

viral assembly

binds viral RNA & cellular factors

31
Q

How can RNA replication be blocked via NS5A mutation?

A

mutate Zn binding site or phosphorylation sites

32
Q

Dimers of NS5A?

A

Close and open structure dimers - different functions?

one involved in replication, other in assembly?

33
Q

What is NS5B?

A

RNA-dependent RNA polymerase

34
Q

Role of NS5B?

A

catalyses production of both neg and pos strands of RNA

35
Q

What does the HCV 3’UTR binds to?

A

NS5A - by Poly U/UC region

NS5B

36
Q

Role of HCV 3’ UTR?

A

initiation of NEGATIVE strand replication

37
Q

What is the HCV 5’ UTR?

A

IRES

binds to ribosome & initiation factors

38
Q

What is the Membranous web of HCV?

A

Double-membraned vesicles

concentrates replication components

protects from innate immune response

39
Q

What is the ratio of NS5B to neg strand RNA?

A

1000:1

why? maybe most NS5B are inactive?

MICR2222 - Medical Virology (13 decks)

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