Hepatitis C Virus 2 Flashcards
JFH-1 replicon origins and use
HCV genotype 2a cloned from patient with fulminant hepatitis
Replicons replicate in Hela/HEk293T cells no need for Huh7
Transfection leads to production of infectious viral particles
Allows examination of Full HCV effect on cell not just translation and polyprotein processing of HCV
HCV cell entry
Multiple proteins involve
6 or 7
Interacts with proteins
GAG, LDL-R, SRB1, CD81, Claudin and occludin
Also interacts with Epidermal growth factor EGRF and related protein within receptor A2 (EphA2) - single TM receptor tyrosine kinases- bind RGF and related Ligands
Niemann-pick C1-like 1 cholesterol absorption receptor (NPC1L1)
- 13 TM receptor for cholesterol uptake
- target for ezetimibe- cholesterol lowering drug
Role of HCV UTRs?
Role in initiation of negative strand replication
Binds NS5A and NS5B
Contains IRES
Highly ordered RNA structure which allows cap independent initiation of translation
Binds to ribosome and initiation factors
HCV can have more efficient RNA translation without switching off the host cell translation
HCV structural proteins
Core-nucleocapsid protein
——nucleic acid binding, associated with ER membrane/lipid droplets
E1/E2- envelope glycoprotein –receptor binding
P7 transmembrane Ion channel (no certaitt is structural protein
Need for HCV signal peptides
Structural proteins are produced as one big polyprotein that need to be cleaves from one another
Core remains on cytosoloc side of ER membrane - signal peptides gets cleaves by an integral protein peptidase in the ER lumen
Core protein later associates and accumulates with lipid droplets
E1 and E2 have trans-membrane domains- form a covalent complex in the ER membrane
E1 and E2 complex retained in ER- does not traffic to cell surface
P7- forms a heptameric ion channel
In the ER membranes
Membrane topology of HCV structural proteins?
E1 and E2 bind to one another
E1 and E2 are in the lumen, core is on the cytoplasmic end of the membrane
–useful during budding (core can be on the inside of the membrane whilst E1/2 are on the outside
HCV non-structural protein 2
NS2 is multiple transmembrane helices
Cleaves itself from NS2-3 precursor
- unusual event in cell
- potential antiviral target –> but nothing has been made to target it yet
Not really required for RNA replication
Probably dimerises
Function in virus assembly and release
Not much is known as subgenomic replicons start from NS3
HCV NS3
Is classic target for antiviral activity
Has protease capability
Once cleaves from NS2, NS3 folds itself and cleaves between other structural proteins (NS4A, 4B, 5A and 5B)
NS4a is a membrane targeting protein that can be cleaved
Good antiviral drug target
NS3 targeting drug
BILB-2061
- oral availability
- initial trials are promising 3 log drop in viral titre after 25h
- dropped after animal cardiac toxicity issues
Boceprevir
Telaprevir
Both of these drugs discontinued due to side affects like anaemia and skin rash
Replaced by second generation forms
HCV NS3/4a
Blocks induction of INF-B expression
Cell usually responds to PAMPS to induce interferon response
–Either TLR3( associated to TRIF) or RIG-1(associated to mitochondria proteins MAVS)
will detect the dsRNA to trigger expression of INF-B(by activating a nuclear localising IRF-3 transcription factor)
NS3/4a recognises a sequence motif in TRIF and it cleaves it–>blocking its activity
NS3/4a also cleaves MAVS
NS3 good antiviral therapy target as you can maintain production of IFNs whilst reducing cleavage of non-structural polyproteins
HCV NS4B
Is a multiple membrane spanning protein
Induces membrane rearrangements –> forming the membranous web
Highly hydrophobic so it is hard to crystalis and examine
HCV NS5A
Membrane surface associated protein
–contains an amphipathic helix that contain hydrophobic A.A on one side and hydrophilic on the other side
Also contains low complexity sequences which gives flexibility
Key component of RNA replication complex
Key role in virus assemble domain 1 and 3 used for virus assembly
Binds viral RNA
Binds to multiple cellular factors 130+proteins
NS5A dimer structure
Forms dimer with two forms
Open and closed form
Probably regulated by phosphorylation as low complexity sequence region 1 has at least 7 serine phosphorylated sites
NS5A is novel drug target name drug against it
Daclatasvir (DCV)
- EC50 between 9-50 picoMolars
- Resistance maps to amphiphatic helix domain
In an infected cell there ~100,000 copies of NS5A. Can be treated with 6 molecule of DCV per cell( really effective)
HCV NS5B
70kDa protein- RNA dependant RNA polymerase
Tail anchored- C terminal membrane spanning helix
Catalysed production of both negative and positive RNA strands
Target for new drugs like Sofosbuvir which was approved by the FDA Dec 2013
Costs 84k for 12 weeks of therapy
