Cellular cofactors affecting hepatitis C virus infection and replication

Abstract

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.antivirals ͉ miR-122 ͉ RNAi ͉ HCVcc-siRNA H epatitis C virus (HCV) has a notable ability to establish persistent infections in Ϸ70% of cases, resulting in 130 million chronically infected people throughout the world (1). This prevalence has spurred considerable interest in the study of HCV-host interactions, on both cellular and molecular levels. The inability to grow HCV in cell culture led some groups to focus on the identification of cellular proteins that interact with individual HCV proteins or RNA elements, resulting in the accumulation of a large number of putative HCV-host interactions. Unfortunately, the significance of most of these with respect to the HCV life cycle is currently unknown (reviewed in ref.2). Over the past 6 years, cell culture systems have been developed that enable the characterization of HCV replication and entry (3-6). This effort recently culminated in the development of cell culture systems that reproduce the entire viral life cycle (7-9). A number of virus-host interactions have been characterized by using these experimental systems. For example, CD81 has been demonstrated to play a role in HCV entry (10-12).Sequence-specific gene silencing of RNAi is ideal for assessing the genetic phenotypes associated with virus-host interactions. We have previously shown that siRNAs are highly effective at silencing either host or viral RNAs in cells that contain replicating HCV, demonstrating th...

Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in Humans: In Vitro and In Vivo Correlations

“…6 Informed written consent was obtained from all patients. HCV RNA levels were determined using the Roche Cobas TaqMan HCV Test, v2.0 (lower limit of quantification, 25 IU/mL; lower limit of detection, 10 IU/mL; Roche, Pleasanton, CA) at baseline and days 1 (2,4,6,8,12,16, and 20 hours post-first dose), 2,3,4,5,7,9,11,14,15,16,17,21, and 28. Viral breakthrough was defined as an HCV RNA increase by at least 0.5 log 10 after HCV RNA nadir while receiving BMS-790052.…”

“…NS5A has also been implicated in the modulation of cellular signaling pathways. 1,2 Because it is required in vivo and in vitro for viral replication and has no known human homologs, NS5A provides an attractive target for therapeutic intervention. 3 BMS-790052 is a potent HCV NS5A replication complex inhibitor, with 50% effective concentration (EC 50 ) values of 9 and 50 pM against genotype 1b and 1a replicons, respectively.…”

879 the Effects of Hepatitis C Virus Core Protein on the Expression of Mir-122 in Vitro

“…no positive correlation was observed between miR-122 level and HCV [22,23]. It may be due to not strong correlation between miR-122 level and HCV RNA level or the different cell line.…”

MicroRNAs in Liver Disease: Bench to Bedside

“…11 However, viral infection and replication does not affect the expression of miR-122. 74 Recently, Marquez et al analyzed the expression of miR-122 and miR-21 in liver biopsy samples of HCV-infected patients and uninfected controls and found miR-122 levels to be inversely correlated with fibrotic stage, ALT, AST, while miR-21 correlated positively. 75 It was speculated that the dysregulation of miR-21 and miR-122 rather than the expression levels, could be related to fibrosis.…”