RESUMO
The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses RNA helicase activity. This enzyme is considered to be involved in viral replication, and is expected to be one of the target molecules of anti-HCV drugs. Previously, we established a high-throughput screening system for HCV helicase inhibitors using the Scintillation Proximity Assay (SPA) system [Kyono, K. et al. (1998) ANAL: BIOCHEM: 257, 120-126]. Here, we show improvement of the preparation method for the HCV NS3/4A complex. Alteration of the expression region led to an increase in protein expression. The partially purified full-length NS3 protein showed higher NS3 protease activity without the cofactor NS4A peptide than the truncated protease domain with the cofactor peptide, suggesting that this protein formed a complex with NS4A. NS3 further purified to homogeneity, as judged on silver staining, remained in a complex with NS4A. Characterization of the helicase activity of this full NS3/4A complex using the SPA helicase assay system revealed that this enzyme preferred Mn(2+), and that the optimal pH was 6.0-6.5. The NS3/4A complex could act on a DNA template but could not unwind the M13DNA/DNA substrate.
Assuntos
RNA Helicases/metabolismo , Proteínas não Estruturais Virais/isolamento & purificação , Sequência de Bases , Cátions Bivalentes/metabolismo , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Nucleotídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Contagem de Cintilação/métodos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The C-terminal two-thirds of nonstructural protein 3 (NS3) of hepatitis C virus (HCV) exhibits RNA-dependent NTPase/helicase activity. This enzyme is considered to be involved in viral replication and is expected to be one of the target molecules of anti-HCV drugs. In a search for NTPase inhibitors specific to HCV, we expressed and purified the truncated NS3 NTPase/helicase domain. Here, we report the characterization of its RNA-dependent ATPase activity. This enzyme preferred Mg(2+) and the optimal pH was 7.0. We further investigated the effects of heavy metal ions on the ATPase activity. The mercuric ion inhibited it significantly, the 50% inhibitory concentration being 49 nM. The fact that the inhibitory profile was competitive and that this inhibition was blocked in the presence of a large excess of cysteine or dithiothreitol, suggested that a cysteine residue in the DECH box was the main target site of mercury.
Assuntos
Adenosina Trifosfatases/química , Mercúrio/farmacologia , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Ligação Competitiva , Cátions , Cistina/química , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Íons , Cinética , Magnésio/química , Metais/química , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.
