Department Of Medical Biotechnology School Of Allied Medicine

IUMS

INHIBITION OF HEPATITIS C VIRUS INFECTION BY NS5A-SPECIFIC APTAMER

Soheila Shokrollahzade

MSc student

Hepatitis C virus (HCV) infects 170 million people worldwide

up to 80% of those infected become chronic infection.

HCV infection can cause chronic hepatitis, liver cirrhosis and even

hepatocellular carcinoma (Laucer and Walker, 2001).

no vaccine available

peginterferon alpha (Peg-IFN-a)-based therapy is the current

treatment for the patients (Hoofnagle and Seeff, 2006).

tenascin-binding aptamer under development by Schering AG for cancer imaging

RNA Aptamer Complexed with Vitamin B12

Anti-VEGFR2 aptamer

Hepatitis C Virus

single positive-strand RNA virus genome: about 9.6 kb in length encodes a single long polyprotein which

is processed into

Structural proteins : core, E1 , E2

Non structural proteins: NS2, NS3, NS4A, NS4B,NS5A and NS5B

Master regulatory of HCV replication and assembly; NS5A protein

Domain 1:

i. an anchorage for connecting the protein to ER membrane.

ii. Contains a zinc binding domain (ZBD) to eliminate the ability of replication

Domain2:

i. interaction with RNA dependant- RNA polymerase

ii. ISRD region (Interferon sensitivity determining region)

Domain 3 :

Viral particle production PDB code:4DMB

Why Aptamers?Aptamers Vs Antibodies

(1) High stability of aptamers

(2) Production of aptamers (synthesis/modification)

(3) Low immunogenicity of aptamers

(4) Variety of target

Expression And Purification Of Hcv NS5A Protein

Western blot analysis of purified NS5A protein. His-tagged NS5A was expressed by IPTG induction in E. coli BL21 (DE3) and then confirmed using mouse anti-His monoclonal antibody (left) or anti-NS5Amonoclonal antibody (right) via western blot

Selective Evolution Of Ligands By Exponential Enrichment (SELEX)

Tom Strachan ,Human molecular genetics

Comparison of the fluorescence of different pools of FITC-labeled DNA binding to NS5A protein or control protein LacZ.

Topmost Sequences Representative The Study

Binding affinity 1:

1. ELONA (Enzyme-Linked Oligonucleotide Assay)Binding affinity of aptamers to recombinant protein NS5A

ELONA (Enzyme-linked Oligonucleotide Assay)

Binding affinity study 2,

The binding affinity of aptamer NS5A-4, NS5A-5 for NS5A protein in lysates of HCV-infected hepatocytes

BINDING AFFINITY STUDY 3,APT3-CO-LOCALIZATION OF NS5A-SPECIFIC APTAMERS WITH NS5A PROTEIN IN THE HCV-INFECTED HEPATOCYTES

NS5A APTAMER DISRUPTS THE INTERACTION OF NS5A WITH CORE

PROTEIN

Interaction of NS5A with core protein is essential for

infectious virus production (Masaki et al., 2008).

Disruption the interaction of NS5A with core protein impairs

infectious virus production (Jiang and Luo, 2012).

Co-immunopercipitation

Immunopercipitation

Co-immunopercipitation

CORE protein in immuno- precipitates with equal amount of NS5A protein in aptamer NS5A-4 or NS5A-5-treated hcv infected Huh7.5 cells was lower than that in library-treated group

INHIBITION OF HCV INFECTION BY NS5A-SPECIFIC APTAMERS …….qPCR ANALYSIS

Domains I And III Of NS5A Protein Are Involved In The Antiviral Effects Of Ns5a-specific Aptamer NS5A-4

Domains I and III of NS5A protein are involved in the antiviral effects of NS5A-specific aptamer NS5A-4