Introduction Hepatitis C Virus Introduction microRNAs Liver-specific MicroRNA-122 displays position dependent function Christiane Brohm 27.10.2008

Hepatitis C Virus (HCV) Profile Family: Flaviviridae Genus: Hepacivirus Species: Hepatitis C virus (6 genotypes) Size: 50-60 nm Genome: (+) ssRNA, ~9.6 kb Prevalence: 130 million patients Therapy: PEG-IFN-a + Ribavirin E1 E2 ss(+) RNA Nucleocapsid (core) Lipid membrane

HCV Genome Organisation

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

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

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.

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

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

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

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

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

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

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

Thank‘s for your attention!