Role of phosphatidylinositol 4-phosphate (PI4P) and its binding protein GOLPH3 in hepatitis C virus secretion

Abstract

Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.

FIGURE 1.

PI4P depletion by hSac1 K2a. A, uninfected Huh7 cells were transfected with pEGFP-hSac1 K2A (green). 3 days post-transfection, cells were fixed and stained for DAPI (cyan), TGN46 (red), and PI4P (blue). B, HCV JC1-infected Huh7 cells were transfected with pEGFP-hSac1 K2A (green). 3 days post-transfection, cells were fixed and stained with antibodies for HCV envelope glycoprotein E2 (cyan), PI4P (red), and TGN46 (blue). Nuclei are demarcated with white circles. hSac1 K2A-expressing cells are marked with white arrowheads.

FIGURE 2.

PI4P depletion affects secretory pathways. A, ssHRP secretion assay. ssHRP-FLAG was co-transfected with control or hSac1 K2A-expressing vector, and the clarified cell supernatants collected at 32, 40, and 48 h post-transfection were assayed for peroxidase activity as described under “Experimental Procedures.” The experiment was done in triplicate, and the data shown are mean ± S.E. ***, p < 0.001; **, p < 0.01 by unpaired t test. B and C, Huh7cells were transfected with hSac1 K2A or a control vector. 2 days post-transfection, culture media were replaced with serum-free media, and the culture supernatants (B) and cell lysates (C) collected 24 h later were assayed for ApoB levels. D and E, densitometric analysis of the Western blots shown in B and C.

FIGURE 3.

Effect of hSac1 K2A-mediated PI4P reduction on HCV replication and secretion. A, JC1-RLuc-2A-infected Huh7.5.1 cells were transfected with either hSac1 K2A or a control vector. Cells were collected at day 3 post-transfection, and cell lysates were assayed for Renilla luciferase reporter activity (A) to measure HCV replication as described under “Experimental Procedures.” B and C, JC1-infected Huh7.5.1 cells were transfected with hSac1 K2A or a control vector. Supernatants and cell lysates were analyzed by FFU assay for extracellular (B) and intracellular (C) infectivity. All experiments were done in triplicate, and the data shown are mean ± S.E. *, p < 0.05 by unpaired t test.

FIGURE 4.

PI4KIIIβ knockdown inhibits HCV secretion and increases intracellular infectivity. A, Huh7.5.1 cells were transfected with either a control or a single or pooled siRNA for PI4KIIIβ, and quantitative RT-PCR performed using PI4KIIIβ-specific primers for quantification of PI4KIIIβ mRNA levels. B, effect of PI4KIIIβ knockdown on HCV replication was assessed by quantitative RT-PCR for HCV RNA using total cellular RNA from JC1-infected Huh7.5.1 cells transfected with control or pool of siRNAs against PI4KIIIβ 72 h post-transfection. C and D, JC1 infected Huh7.5.1 cells were transfected with either a control or a pool of siRNAs (1 and 3), and 72 h post-transfection, the infectivity of cell culture supernatants (C) and cell lysates (D) determined by FFU assay as described under “Experimental Procedures.” All experiments were done in triplicate, and the data shown are mean ± S.E. **, p < 0.01; *, p < 0.05 by unpaired t test.

FIGURE 5.

Effect of GOLPH3 knockdown on HCV replication and secretion. JC1-infected Huh7.5.1 cells were transfected with either a control or siRNA specific to GOLPH3. Cell lysates (A) were blotted for GOLPH3 and MYO18A, and calnexin was used as a loading control. 3 days post-transfection total RNA was isolated from culture supernatant (B) and cell lysate (C), and HCV RNA levels evaluated by quantitative RT-PCR as described under “Experimental Procedures.” D and E, JC1-infected Huh7.5.1 cells were transfected with either a control siRNA or siRNAs to GOLPH3 or MYO18A. 3 days post-transfection, lysates and supernatants were collected, and extracellular infectivity (D) and intracellular infectivity (E) were determined by FFU assay. All experiments were done in triplicate, and the data shown are mean ± S.E. **, p < 0.01; *, p < 0.05 by unpaired t test.

FIGURE 6.

Effect of HCV infection on the Golgi network. HCV-infected and uninfected cells are stained with various Golgi-specific antibodies. Cells were also stained with antibodies specific to PI4P, GOLPH3, and PI4KIIIβ. Nuclei were counterstained with DAPI. Infected cells are marked with white arrows, whereas uninfected cells are denoted with an X.