NS5B

About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.

A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle.

Abstract: Globally, approximately 170 million people (representing approximately 3% of the population worldwide), are infected with hepatitis C virus (HCV) and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs)-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B) on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinker™ glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL−1 to 1 ng mL−1 range with a detection limit of 1 ng mL−1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well.

Immune responses and immunity in hepatitis C virus infection

Abstract: The hepatitis C virus is an enveloped, positive-strand RNA virus, approximately 50 nm in diameter2,3 and similar in genomic structure to Flaviand Pestiviridae. Its genome consists of approximately 9600 nucleotides and contains a single open reading frame encoding a polyprotein of approximately 3000 amino acids. Both host and viral proteases cleave this polyprotein into three structural proteins—core, E1, and E2—and six nonstructural proteins—NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The function of p7, located between structural and nonstructural proteins is currently not known (for a detailed review of the HCV genome structure, replication and proteins functions, see references 4 and 5). HCV enters a susceptible host either directly, through transfusion of contaminated blood products or injection with contaminated needles, or, less efficiently, by crossing over an epithelial barrier, as exemplified by perinatal or sexual transmission. The virus reaches the liver via the hepatic artery or the portal vein and enters hepatocytes, its preferred site of replication. Sharp serum alanine aminotransferase (ALT) elevations may occur between 10 and 16 weeks after infection, but acute hepatitis is rarely diagnosed, because the majority of patients are not icteric and do not develop any symptoms other than fatigue and mild pain in the right upper abdomen. Circulating HCV-specific T cells have been demonstrated as early as 3–4 weeks after infection, whereas HCV-specific antibody responses occur much later, between 7 and 31 weeks after infection.6

Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

Abstract: Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCE While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the Δ1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.