PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

Abstract

Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly. IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.

Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly

“…Perilipin-2 expression is required for trafficking of core and NS5A to LDs and for the formation of HCV particles (15). We also showed that overexpression of PACSIN2 promotes NS5A and core interaction, which facilitates HCV particle assembly (16).…”

“…However, the proportion of intracellular and extracellular infectivity was not altered in cortactin knockdown cells (Fig. 5E), indicating that cortactin might be involved in the assembly step but not the release step of the HCV life cycle (16,(42)(43)(44).…”

“…Protein interplay between NS5A and core plays a crucial role in HCV particle production (12). We previously reported that PACSIN2 potentiates protein interplay between NS5A and core to promote HCV assembly (16). To investigate how cortactin was involved in HCV assembly, we assessed the effect of cortactin on NS5A-core interaction.…”

“…Preparation of infectious virus. Cell culture-grown HCV (HCVcc) was generated as described previously (16). The monolayer of Huh7.5 cells was washed twice in phosphate-buffered saline (PBS), trypsinized, and centrifuged at 850 rpm for 3 min (16).…”

The Beginning of Ending Hepatitis C Virus: A Summary of the 26th International Symposium on Hepatitis C Virus and Related Viruses

“…Nguyen et al sought to investigate the role of protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) interactions with HCV NS5A protein. Their data indicate that HCV exploits the pro-viral cellular function of PACSIN2 to promote viral assembly [11]. Takeo et al examined the potency of HCV NS3-specific nanobodies in HCV-infected cells and found that HCV NS3-specific nanobodies do not affect HCV RNA replication, but potently inhibit virus assembly at the stage of nucleocapsid envelopment.…”