Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly

<div><p>Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to <em>trans</em>-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.</p> </div

AAK1 and GAK regulate core-AP2M1 binding and are essential for HCV assembly.

<p>A. Regulatory mechanisms of AP2M1 binding to host cargo proteins harboring YXXΦ signals. B. Binding of core to wild type and T156A AP2M1 mutant by PCAs (black) and microfluidics (white). (C–E) Huh-7.5 were transfected with plasmids encoding WT or T156A AP2M1 mutant and electroporated with J6/JFH(p7-Rluc2A) 48 hr posttransfection. C. Cellular viability by alamarBlue-based assays at 48 hr posttransfection relative to WT AP2M1 control. D. HCV RNA replication in cells overexpressing WT or T156A AP2M1 mutant by luciferase assays at 6 hr (black) and 72 hr (white) postelectroporation with J6/JFH(p7-Rluc2A). E. Extracellular (black) and intracellular (white) infectivity by luciferase assays in naive Huh-7.5 cells infected with supernatants or cell lysates derived from the indicated cells, respectively, relative to WT control. (F–G) Huh7.5 cells were transfected with the corresponding siRNAs. F. Ratio of AAK1 (left) or GAK (right) to S18 RNA in these cells relative to NT sequences by qRT-PCR. G. Quantitative Western analysis. Numbers represent AAK1 (top) or GAK (bottom) to actin protein ratios relative to NT control. H. Core-AP2M1 binding by PCAs in Huh-7.5 cells depleted for AAK1 or GAK by siRNAs. Y axis represents luminescence ratio (the average luminescence signal detected in cells transfected with Gluc1-AP2M1 and Gluc2-core divided by the average of luminescence measured in NT cells transfected with Gluc1-AP2M1 and an empty Gluc2 vector with those transfected with Gluc2-core and an empty Gluc1 vector) relative to NT control. (I–K) AAK1 or GAK depleted cells were electroporated with J6/JFH(p7-Rluc2A). I. Cellular viability by alamarBlue-based assays in depleted cells relative to NT control. J. HCV RNA replication in these cells by luciferase assays at 6 hr (black) and 72 hr (white) postelectroporation. K. Extracellular (black) and intracellular (white) infectivity by luciferase assays in naive Huh-7.5 cells infected with supernatants or cell lysates derived from the indicated cells, respectively, relative to NT control. L. Core binding to AAK1 and GAK by PCAs. Y axis represents luminescence ratio relative to core-AP2M1 binding. Data represent means and s.d. (error bars) from at least two experiments in triplicates. RLU is relative light units. * p<0.05, ** p<0.01, *** p<0.001.</p

Core binds AP2M1 in cells and in the context of HCV infection.

<p>A. Schematics of the PCAs format. A and B represent prey and bait proteins and GLuc1/2 are fragments of <i>Gaussia</i> luciferase. B. Cells were cotransfected with combinations of plasmids indicated below the graph with those indicated in the legend. Y axis represents luminescence relative to the core-AP2M1 signal. The banded bar on the right represents AP2M1 binding to the host cargo protein TFR. C. Core-AP2M1 binding in the presence of free AP2M1, core or NESI. Y axis represents luminescence ratio (the average luminescence signal detected in cells transfected with Gluc1-AP2M1 and Gluc2-core divided by the average of luminescence measured in control wells transfected with Gluc1-AP2M1 and an empty Gluc2 vector with those transfected with Gluc2-core and an empty Gluc1 vector) relative to core-AP2M1 binding in the presence of empty PUC19. D. Immunoprecipitations (IPs) in membranes of HCV infected cells. Left panels: Anti-AP2M1 antibodies or IgG were used for IP. Membranes were immunoblotted (IB) with anti-phospho-AP2M1, anti-AP2M1, anti-core, and anti-actin antibodies. Cal-A represents calyculin-A. Right panels: Anti-core antibodies or IgG were used for IP. Membranes were immunoblotted (IB) with anti-core, anti-AP2M1, and anti-actin antibodies. E. Representative confocal IF microscopy images of AP2M1 and core in Huh-7.5 cells 3 days postelectroporation with J6/JFH HCV RNA. Data represent means±s.d. (error bars) from three independent experiments in triplicates (n>20 in E). * p<0.05, ** p<0.01, *** p<0.001.</p

Pharmacological inhibition of core-AP2M1 binding and HCV assembly.

<p>A. AP2M1 regulators and the discovered inhibitors. B. The inhibitors' Kds of binding to AAK1 or GAK <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002845#ppat.1002845-Karaman1" target="_blank">[78]</a>, IC50s for these compounds effect on core-AP2M1 binding, and EC50s for their effect on extracellular infectivity, intracellular infectivity, and viral infection with cell culture grown HCV (HCVcc). C. Inhibition of core-AP2M1 binding by the compounds measured by PCAs. D–G. Huh-7.5 cells electroporated with J6/JFH(p7-Rluc2A) were treated daily with either erlotinib, sunitinib or PKC-412 for 3 days. Supernatants and cell lysates were harvested at 72 hr and used to inoculate naive Huh-7.5 cells. Dose response curves of the inhibitors' effects on extracellular (D) and intracellular (E) infectivity relative to untreated controls. These compounds had no effect on HCV RNA replication (F) or cellular viability (G) by luciferase and AlamarBlue-based assays, respectively (GNN is a replication-defective polymerase mutant). H. The effect of the inhibitors on AP2M1 phosphorylation by Western analysis of cell lysates harvested following electroporation with J6/JFH(p7-Rluc2A) and treatment with the compounds in the presence of Calyculin A (Cal-A). Representative membranes blotted with anti-phopho-AP2M1 (p-AP2M1) and anti-actin antibodies, and quantitative analysis from 3 experiments are shown. Y axis represents pAP2M1/actin protein ratio relative to untreated controls. I. The inhibitors' effect on viral infection (black) and cellular viability (grey) in cells infected with HCVcc following 72 hr of daily treatment relative to untreated controls. Data represent means and s.d. (error bars) from at least three experiments in triplicates. RLU is relative light units. * p<0.05, ** p<0.01, *** p<0.001.</p

Disruption of core-AP2M1 binding abolishes recruitment of AP2M1 to LD and alters the sub-cellular localization of core and its colocalization with E2.

<p>Quantitative confocal immunofluorescence (IF) analysis of the sub-cellular localization of core and AP2M1 and core-E2 colocalization in Huh-7.5 cells. A. A representative merged image of endogenous AP2M1 (blue) and the LD marker, Bodipy (green), in naive Huh-7.5 cells. B–D. Merged images of Huh-7.5 cells infected with J6/JFH HCV stained for core (red), the LD marker, Bodipy (green), and AP2M1 (blue). E. Percent colocalization of the indicated signals in naive (white) or infected (black) cells by a quantitative colocalization analysis of A–D. F. A four channel merged image. The yellow arrows in the inset indicate colocalization of core and AP2M1 to LD. G–J. Representative merged images and quantitative colocalization analysis of AP2M1 (red) and the lipid marker, LipidTOX (blue), in Huh-7.5 cells transfected with plasmids expressing AP2M1-mCherry alone (G) or AP2M1-mCherry with WT core (H) or core Y136A mutant (I). Core expression (green) in the cells shown in panels H and I is demonstrated in the respective bottom panels. (K–P) Representative merged images and quantitative colocalization analysis of core (red) and: K. Bodipy (green), demonstrating increased localization of core to LD in Huh-7.5 cells electroporated with J6/JFH HCV RNA harboring the Y136A core mutation (right panel) compared with WT core (left panel). L. Bodipy (green) in control (NT) cells (left panel) or AP2M1 depleted (right panel) Huh-7.5 electroporated with J6/JFH HCV RNA, showing a dramatic localization of core to LD in AP2M1 depleted cells. M. TGN46 (green), demonstrating decreased localization of core to TGN in Huh-7.5 cells electroporated with J6/JFH RNA harboring the Y136A core mutation (right panel) compared with WT core (left panel). N. TGN46 (green) in control (NT) cells (left panel) or AP2M1 depleted (right panel) Huh-7.5 electroporated with J6/JFH HCV RNA, showing decreased localization of core to TGN in AP2M1 depleted cells. O. E2 (green), demonstrating decreased colocalization of core and E2 in Huh-7.5 cells electroporated with J6/JFH RNA harboring the Y136A core mutation (right panel) compared with WT core (left panel). P. E2 (green) in control (NT) cells (left panel) or AP2M1 depleted (right panel) Huh-7.5 electroporated with J6/JFH HCV RNA, showing decreased colocalization of core and E2 in AP2M1 depleted cells. Representative images at ×60 magnification are shown. Graphs represent quantitative colocalization analysis of Z stacks using Manders' coefficients. Values indicate mean M2 values represented as percent colocalization (the fraction of green intensity that coincides with red intensity or in the case of Figures G–I, the fraction of blue intensity that coincides with red intensity) ± s.d. (error bars); n = 10–15. * p<0.05, ** p<0.01, *** p<0.001.</p

AP2M1 depletion inhibits HCV assembly.

<p>A. AP2M1 protein levels by quantitative Western analysis in stable clones harboring shRNA lentiviral constructs targeting the AP2M1 gene and a non-targeting (NT) sequence. A representative membrane and combined data from three independent measurements are shown. Y axis represents AP2M1 to actin protein ratio relative to NT control. B. AP2M1/S18 RNA ratio by qRT-PCR in selected stable clones relative to NT control. C. The indicated clones were electroporated with J6/JFH(p7-Rluc2A). HCV RNA replication in these clones by luciferase assays at 9 hr (white) and 72 hr (black) postelectroporation. D. Extracellular infectivity measured by luciferase assays in naive cells inoculated with supernatants derived from the various stable cell clones. E. Intracellular infectivity by luciferase assays in naive Huh-7.5 cells infected with clarified cell lysates derived from the electroporated cells. F. Intra- and extracellular infectivity titers measured by limiting dilution assays. TCID₅₀ is 50% tissue culture infectious dose. G. Viral RNA release into the culture supernatant at 72 hr postelectroporation measured by qRT-PCR. H. HCV core protein release into the culture supernatant at 72 hr postelectroporation, as determined by ELISA. I. Infectious virus production relative to NT control (top panel) and levels of AP2M1 protein by a Western blot analysis (bottom panel) in cells concurrently transduced with lentiviruses expressing shAP2M1 and shRNA resistant WT AP2M1 cDNA (AP2M1-WT). Means and s.d. (error bars) of results from at least three independent experiments are shown. RLU is relative light units. * p<0.05, ** p<0.01, *** p<0.001.</p

Core harbors a YXXΦ motif and binds AP2M1.

<p>A. Schematics of core. The location of the identified motif is indicated. B–C. Consensus sequences of YXXΦ motifs from representative human (B) and viral (C) proteins. D. The consensus sequence of all HCV isolates, clones used in this study, and engineered core mutants. E. Fluorescent images from a microfluidic chip and schematics. Left: AP2M1-V5-his was anchored to the device surface via its interaction with anti-His antibodies and labeled with anti-V5-FITC antibodies. Middle: T7-tagged core or NS3 were incubated with surface bound AP2M1 and labeled with anti-T7-Cy3 antibodies. Interactions were trapped mechanically by MITOMI. Cy3 signal representing bound viral protein is shown following a wash. Right: an overlay of the Cy3 and FITC signals, representing bound viral prey to human bait ratio. F. <i>In vitro</i> binding curves of core-T7 and NS3-T7 to surface bound AP2M1. Y axis represents bound viral protein to surface bound AP2M1 ratio.</p

Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents

Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo­[5,4-<i>b</i>]­pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson’s disease)