Abstract
The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy.
Highlights
ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1), known as ARP17, is a membrane-associated 25-kDa GTPase
We demonstrated that silencing of ADP-ribosylation factor-related protein 1 (ARFRP1) impaired hepatitis C virus (HCV) RNA and protein expressions, and subsequent HCV infectivity
Exogenous expression of the small interfering RNA (siRNA)-resistant mutant ARFRP1 recovered extracellular HCV RNA level (Fig. 1J, lane 4 versus lane 5) in ARFRP1-knockdown cells. All these data suggest that ARFRP1 is required for HCV propagation
Summary
ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1), known as ARP17, is a membrane-associated 25-kDa GTPase. Knockout of ARFRP1 gene in mice resulted in embryonic lethality and apoptosis in ectodermal cells[18]. ARFRP1 is essential for cell survival[18] and regulates the growth of LDs7,22. We demonstrated that silencing of ARFRP1 impaired HCV RNA and protein expressions, and subsequent HCV infectivity. Knockdown of ARFRP1 significantly reduced HCV-mediated LD growth. We further showed that SNAP23 protein, a downstream effector of ARFRP1 which has been known to be required for LD assembly, was required for HCV production. Our study provides the first evidence that HCV regulates ARFRP1 together with SNAP23 for LD growth to facilitate viral propagation
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