Mechanism of interferon resistance of hepatitis C virus RNA

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Hepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and possesses multiple functions. Some of functions may be mediated by the direct interaction with RNA.In order to clarify the interaction between the core protein and nucleic acids, we showed the property of glutathione S-transferase (GST)-core protein by activity gel in which protein is immobilized. It has been very difficult to electrophoretic mobility shift assay (EMSA) using the core protein due to extremely basic character. Therefore, the plasmid was constructed, encoding neutral fusion protein composed of GST, 47 amino acids-carboxyl terminal region of HCV nonstructural 5A protein (NS5Ac) which is acidic, and the core protein between amino acids 1 and 90 (C<@D21-90@>D2). The recombinant protein named GST-NS5Ac-C<@D21-90@>D2 was purified with homogeneity and used for biochemical analysis including EMSA.When 24-mer oligonucleotide corresponding to (CU<@D1n@>D1) repeat region of 3' terminal region of HCV was used … More as a probe, EMSA revealed that the GST NS5Ac-C<@D21-90@>D2 bound strongly to double-stranded RNA (dsRNA) but not to single-stranded RNA (ssRNA). The GST-NS5Ac-C<@D21-90@>D2 protein also strongly bound to dsDNA and DNA/RNA heteroduplex. Competition assays indicated that the order of affinity was dsRNA(]SY.di-substituted left.[) dsDNA (]SY.di-substituted left.[)(]SY.di-substituted left.[) ssRNA or ssDNA.The GST-NS5Ac-C<@D21-90@>D2 protein efficiently retarded some ssRNAs such as 5' non-coding region of HCV, and RNA including 5' portion of the HCV core gene although the affinity for ssRNA was lower than that for corresponding dsRNA, suggesting that the binding of the core protein to ssRNA is sequence-dependent manner, while the binding of the core to dsRNA is sequence-independent manner. The binding affinity of GST-NS5Ac-C<@D21-90@>D2 for dsRNA was comparable to that of interferon-inducible dsRNA dependent protein kinase (PKR) in vitro, and the equilibrium constant between GST-NS5Ac-C<@D21-90@>D2 and dsRNA was estimated about 1.5 * 10<@D1-8@>D1 M.When the PKR was incubated with the core fusion protein, PKR activity was inhibited depending on the concentration of GST-NS5Ac-C<@D21-90@>D2 protein but not GST-NS5Ac. These results suggest that the core protein potentiates to inhibit the PKR activity. Together with recent finding of the NS5A function, we propose the hypothesis that two HCV gene products, the core protein and NS5A protein, act in a synergistic manner and inhibit PKR activity to preclude the shutoff of protein synthesis. Less