Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX

Although picornaviruses are conventionally considered ‘nonenveloped’, members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX

A Study of T Cell Tolerance to the Tumor-Associated Antigen MDM2: Cytokines Can Restore Antigen Responsiveness, but Not High Avidity T Cell Function

BACKGROUND: Most tumor-associated antigens (TAA) currently used for immunotherapy of cancer are also expressed in normal tissues, which may induce tolerance and impair T cell-mediated immunity. However, there is limited information about how physiological expression in normal tissues alters the function of TAA-specific T cells. METHODOLOGY/PRINCIPAL FINDINGS: We used a T cell receptor transgenic model to study how MDM2 expression in normal tissues affects the function of T cells specific for this TAA that is found at high levels in many different types of tumors. We found that some MDM2-specific T cells escaped thymic deletion and persisted in the peripheral T cell pool. When stimulated with antigen, these T cells readily initiated cell division but failed to proliferate and expand, which was associated with a high rate of apoptosis. Both IL-2 and IL-15 efficiently rescued T cell survival and antigen-specific T cell proliferation, while IL-7 and IL-21 were ineffective. Antigen-stimulated T cells showed impaired expression of the effector molecules CD43, granzyme-B and IFN-γ, a defect that was completely restored when T cells were stimulated in the presence of IL-2. In contrast, IL-15 and IL-21 only restored the expression of CD43 and granzyme-B, but not IFN-γ production. Finally, peptide titration experiments with IL-2 rescued T cells indicated that they were of lower avidity than non-tolerant control T cells expressing the same TCR. CONCLUSIONS/SIGNIFICANCE: These data indicate that cytokines can rescue the antigen-specific proliferation and effector function of MDM2-specific T cells, although this does not lead to the recovery of high avidity T cell function. This study sheds light on possible limitations of immunotherapy approaches that target widely expressed TAA, such as MDM2

Nanomolar-potency small molecule inhibitor of STAT5 protein

© 2014 American Chemical Society. We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a, possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1, and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials

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

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

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 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