1. Introduction Hepatitis C Virus
Introduction microRNAs
Liver-specific MicroRNA-122 displays position dependent function
Christiane Brohm

2. Hepatitis C Virus (HCV) Profile
Family: Flaviviridae
Genus: Hepacivirus
Species: Hepatitis C virus
(6 genotypes)
Size: nm
Genome: (+) ssRNA, ~9.6 kb
Prevalence: 130 million patients
Therapy: PEG-IFN-a + Ribavirin E1 E2
ss(+) RNA
Nucleocapsid (core)
Lipid membrane

3. HCV Genome Organisation

4. MicroRNA Mechanism of Action
▪ 21-22nt RNA molecule
▪ Expression of microRNAs in wide range of eukaryotic organisms
▪ Inhibition of mRNA expression by translation repression or mRNA cleavage

5. Liver-specific microRNA: miR-122
RISC complex
miR-122
Upregulation of HCV Replication

6. Position-Dependent Function for a Tandem MicroRNA
Aim of the study
Analysis of miR-122 function by binding either the 5‘NTR or 3‘NTR.
Importance of the location of the miR-122 binding site for mRNA regulation.
▪ 5‘NTR: upregulative function, increased viral RNA amount
▪ 3‘NTR: downregulative function, decreased viral RNA amount
Identification of an additional binding site and it‘s effect
on reporter-gene expression.

7. 5‘ end insertion does not affect protein synthesis
Insertion of complete HCV 5‘NTR in reporter mRNA.
After sequestration of miR-122 no downregulation of protein synthesis
Synthetic miR-122 did not affect reporter-gene expression in contrast
to effects seen with replication-competent HCV genomes.
Huh7 cells
pHCV.Luc +
Oligonucleotides
reporter gene assay

8. 3‘ end insertion leads to decreased protein synthesis
Insertion of miR-122 binding site at the 3‘ end.
After sequestration of miR-122 upregulation of protein synthesis
Synthetic miR-122 expression induced decreased reporter-gene expression

9. Second, adjacent binding site for miR-122
Second copy of binding site was inserted in 3‘ end of reporter mRNA.
Binding of miR-122 to tandem sites induces a stronger effect on reporter gene expression
Mutation of one binding site displays reduced effects
Mutation of both sites can only compensated by second site mutations in miR-122
→ Tandem sites mediate translational repression

10. Examination of functional role for the second binding site in HCV gene expression
Combination of mutations in either one or both binding sites in a
replication-competent HCV genome.
→ miR-122 interaction at both sites is required for efficient HCV RNA accumulation

11. Can miR-122 function be substituted by other microRNAs?
Exchange of miR-122 binding site with miR-21 binding site in 5‘NTR.
→ miR-122 complexes have specialized functions in HCV replication or
→ Substitution of binding site affects sequences essential for genome amplification

12. Role of spacer sequences surrounding binding sites
Do they have an effect on miR-122 binding or viral genome amplification?
→ Spacer regions (14nt) are highly conserved among HCV genotypes
→ Contribution to the formation of a replication-competent RNA structure

13. Summary
- Insertion of miR-122 binding site in 5‘ end of reporter genomes did not lead to altered protein synthesis.
- Whereas 3‘ end insertion induces decreased reporter gene activity, reminiscent of the outcome of a
typical microRNA-mRNA interaction
- Genetic evidence for presence of 2 functional miR-122 binding sites
- Regulation of protein synthesis is mediated by translational repression since mRNA amount were not reduced
- Both miR-122 binding sites are required for HCV RNA accumulation in replication-competent genomes
- miR-122 function can not be substituted by other microRNAs
- Spacer sequences are highly conserved and contribute to the formation of a
replication-competent RNA structure

14. Thank‘s for your attention!