Abstract
BackgroundHepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone.ResultsThe antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA.ConclusionRecombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.
Highlights
Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide
We showed that intracellular expression of this recombinant antibody clone from a plasmid vector inhibits helicase activity and replication of HCV 1b virus [30]
Cell lines and plasmid clones Huh-7.5 cells were obtained from the laboratory of Dr Charles M Rice (Center for the Study of Hepatitis C, The Rockefeller University, New York) and cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, San Diego, CA) with high glucose supplemented with non-essential amino acids, sodium pyruvate and 5% fetal bovine serum
Summary
Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. A single large polyprotein of 3010 amino acids is translated from the long open reading frame (ORF) encoded within the viral RNA genome. This large protein is cleaved into 10 different individual proteins by the combined action of the cellular and viral proteases. The remaining non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) are essential for replication of HCV positive and negative strand RNA. Among these non-structural proteins, NS3 is the viral protease and NS5B is the viral polymerase. There are large numbers of HCV inhibitors in the clinical developments targeting these two proteins and these new drugs in combination may improve the treatment of chronic HCV infection [6]
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