Hepatitis C Virus NS5A Hijacks ARFGAP1 To Maintain a Phosphatidylinositol 4-Phosphate-Enriched Microenvironment

Abstract

Phosphatidylinositol 4-phosphate (PI4P) is well known to be upregulated during hepatitis C virus (HCV) replication. The role of PI4 kinases in HCV has been extensively investigated. Whether the PI4P phosphatase Sac1 is altered by HCV remains unclear. Here, we identified ARFGAP1 to be a novel host factor for HCV replication. We further show that Sac1 interacts with ARFGAP1 and inhibits HCV replication. The elevation of PI4P induced by HCV NS5A is abrogated when the coatomer protein I (COPI) pathway is inhibited. We also found an interaction between NS5A and ARFGAP1. Furthermore, we identified a conserved cluster of positively charged amino acids in NS5A critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. Our data demonstrate that ARFGAP1 is a host factor for HCV RNA replication. ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the site of HCV replication to maintain high levels of PI4P. Our findings provide an additional mechanism by which HCV enhances formation of a PI4P-rich environment. IMPORTANCEPI4P is enriched in the replication area of HCV; however, whether PI4P phosphatase Sac1 is subverted by HCV is not established. The detailed mechanism of how COPI contributes to viral replication remains unknown, though COPI components were hijacked by HCV. We demonstrate that ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the HCV replication area to maintain high-level PI4P generated by NS5A. Furthermore, we identify a conserved cluster of positively charged amino acids in NS5A, which are critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. This study will shed mechanistic insight on how other RNA viruses hijack COPI and Sac1. H epatitis C virus (HCV) is a major cause of chronic liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma, infecting about 170 million people worldwide (1). Treatment of patients with a combination of pegylated interferon and ribavirin produces only a sustained virological response in about 50% of patients and often produces serious side effects. Direct translation of the HCV genome gives rise to a polyprotein precursor, which can be further processed by host and viral proteases into structural proteins and nonstructural proteins. Nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B are necessary and sufficient for RNA replication (2).A hallmark of HCV replication is the formation of a membranous web which is induced mainly by NS4B (3). Recent studies have shown that NS5A plays an essential role for maintaining membranous web integrity by activating PI4 kinase type III alpha (PI4KA) to elevate phosphatidylinositol 4-phosphate (PI4P) during HCV infection (4-8). Whether the host transport pathway is involved in the PI4P generation by NS5A is unknown.Sac1 is the key phosphatase that dephosphorylates PI4P (9). Previous work has suggested that Sac1 is a coatomer protein I (COPI) cargo which contains a KXKXX motif (10). Key factors in the COPI pathway, in...

Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport

“…PI4P is upregulated and essential during HCV infection . PI4P enrichment at viral replication sites is established by a dual‐mechanism that includes PI4K recruitment to promote PI4P production and Sac1 displacement to slow PI4P turnover . The HCV nonstructural protein 5a (NS5A) is responsible for PI4P synthesis by recruiting PI4KIIIα and stimulating its activity at viral replication sites .…”

“…In addition, NS5A recruits the Arf1 GTPase‐activating protein (Arf1‐GAP), which is known to contribute to cargo sorting and COPI vesicle biogenesis . Because Sac1 is a COPI cargo protein, NS5A recruitment of Arf1‐GAP probably displaces Sac1 from the Golgi, allowing increased PI4P at viral replication sites . Thus, Sac1 and its ability to turn over PI4P normally inhibit HCV replication.…”

“…[192][193][194][195] PI4P enrichment at viral replication sites is established by a dualmechanism that includes PI4K recruitment to promote PI4P production and Sac1 displacement to slow PI4P turnover. 196 The HCV nonstructural protein 5a (NS5A) is responsible for PI4P synthesis by recruiting PI4KIIIα and stimulating its activity at viral replication sites. 193,194,197,198 In addition, NS5A recruits the Arf1 GTPase-activating protein (Arf1-GAP), 196 which is known to contribute to cargo sorting and COPI vesicle biogenesis.…”

Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides

“…An alternative interpretation is that DCV might inhibit other NS5A functions associated with maintaining PI4P pools, such as binding ArfGAP1, which was reported to influence PI4P levels by removing the lipid phosphatase Sac1 from replication compartments [47]. The ArfGAP1 inhibitor, QS11, inhibits HCV replication and reduces the level of PI4P in NS5A expressing cells.…”

Retromer localizes to autophagosomes during HCV replication

“…In the immunofluorescence microscopy assay, Cells seeded onto glass coverslips were washed with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde in PBS buffer for 5 min at room temperature. Fixed cells were stained as previously described (Li et al, 2014). The antibody against LC3B used for immunofluorescence and immunoblot was from Cell Signaling Technology (Danvers, MA, USA) (catalogue No.…”