Abstract
Hepatitis C virus (HCV) is highly dependent on host proteins for its own propagation. By transcriptome sequencing (RNA-Seq) analysis, we identified 30 host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected and characterized ankyrin repeat domain 1 (ANKRD1). Here, we showed that protein expression of ANKRD1 was up-regulated in HCVcc-infected cells. We further showed that protein expression level of ANKRD1 was increased by nonstructural 5A (NS5A) protein. ANKRD1 specifically interacted with NS5A both in vitro and coimmunoprecipitation assays. Protein interaction was mediated through the domain II of NS5A and the C-terminal region of ANKRD1. Promoter activity of ANKRD1 was also increased by NS5A protein. Moreover, up-regulation of ANKRD1 expression was mediated through alteration in intracellular calcium homeostasis and ER stress in HCVcc-infected cells. We showed that silencing of ANKRD1 impaired HCV propagation without affecting HCV replication. By using HCV-like infectious particle (HCV-LP), we demonstrated that HCV single-cycle infection was drastically impaired in ANKRD1 knockdown cells. Finally, we verified that ANKRD1 was required for HCV entry. These data suggest that HCV coopts ANKRD1 for its own propagation and up-regulation of ANKRD1 may contribute to HCV-mediated liver pathogenesis.
Highlights
ankyrin repeat domain 1 (ANKRD1), known as cardiac ankyrin repeat protein (CARP), is a pleiotropic functional protein belong to a conserved family of muscle ankyrin repeat protiens[8,9]
To investigate if transcriptional level of ANKRD1 was regulated by Hepatitis C virus (HCV) infection, Huh7.5 cells were transfected with luc reporter plasmid consisting of − 2000 nt to ± 25 nt of ANKRD1 promoter and infected with Jc1
By RNA-Seq analysis, we identified ANKRD1 which was highly expressed in HCVcc-infected cells
Summary
ANKRD1, known as cardiac ankyrin repeat protein (CARP), is a pleiotropic functional protein belong to a conserved family of muscle ankyrin repeat protiens[8,9]. ANKRD1 is discovered as a novel cytokine-inducible nuclear protein in endothelial cells[10,11]. ANKRD1 is expressed at the highest levels in skeletal muscle and heart where they are localized to the I band of the sarcomere through binding to titin and myopalladin[12]. We demonstrated that protein expression of ANKRD1 was up-regulated in HCVcc-infected cells. We further showed that ANKRD1 expression level was increased by NS5A. Promoter activity of ANKRD1 was increased by NS5A protein. By single-cycle infection assay using HCV-like infectious particle (HCV-LP), we showed that HCV propagation was drastically impaired in ANKRD1 knockdown cells. We demonstrated that ANKRD1 was involved in the entry step but not binding step during HCV infection. These data suggest that ANKRD1 may represent a novel entry factor required for HCV infection
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