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Iron is crucial to the regulation of the host innate immune system and the outcome of many infections. Hepatitis C virus (HCV), one of the major viral human pathogens that depends on iron to complete its life cycle, is highly skilled in evading the immune system. This study presents the construction and validation of a physiologically relevant triple-cell co-culture model that was used to investigate the input of iron in HCV infection and the interplay between HCV, iron, and determinants of host innate immunity. We recorded the expression patterns of key proteins of iron homeostasis involved in iron import, export and storage and examined their relation to the iron regulatory hormone hepcidin in hepatocytes, enterocytes and macrophages in the presence and absence of HCV. We then assessed the transcriptional profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10) under normal or iron-depleted conditions and determined how these were affected by infection. Our data suggest the presence of a link between iron homeostasis and innate immunity unfolding among liver, intestine, and macrophages, which could participate in the deregulation of innate immune responses observed in early HCV infection. Coupled with iron-assisted enhanced viral propagation, such a mechanism may be important for the establishment of viral persistence and the ensuing chronic liver disease.

During HCV infection, the multifunctional core and NS5A viral proteins regulate the iron‐regulatory hormone hepcidin expression antagonistically. While hepcidin is enhanced by core‐mediated activation of BMP/SMAD and STAT3 pathways, it is down‐regulated by NS5A independently of these factors. Herein, we show that NS5A disturbs the MTF‐1/Zn2+/hepcidin positive regulation axis, by mopping up Zn2+, thereby preserving the structural integrity of NS5A dimers., Hepcidin, a 25‐amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron‐regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down‐regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A‐expressing stable cell line, which also exhibited down‐regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well‐characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A‐driven reduction of hepcidin levels, we ruled out any NS5A‐induced alterations in the expression of the well‐known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF‐1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The HCV viral particle is composed of a capsid containing the genome, surrounded by an endoplasmic reticulum (ER)-derived lipid bilayer where E1 and E2 are assembled as heterodimers. However, different forms of viral particles have been identified in the serum of HCV-infected patients, including non-enveloped particles. Previous reports have demonstrated that HCV non-enveloped capsid-like particles (HCVne) can be generated by HCV core protein sequence. This sequence possesses a highly conserved ΥΧΧΦ motif and distal di-leucine motifs that confer primary endocytosis signals, enabling HCVne to enter hepatic cells via clathrin-mediated endocytosis. Although HCV core's primary function is to encapsidate the viral genome, it also interacts with a variety of cellular proteins in order to regulate host cell functions such as gene transcription, lipid metabolism, apoptosis and several signaling pathways. In this report, we demonstrate that the YXXΦ motif of HCV core protein is crucial for the architectural integrity of the particulate form of HCVne. Moreover, we show that the YXXΦ motif in the HCV core sequence plays a pivotal role in the signaling events following HCVne clathrin-mediated endocytosis by inducing the AP-2 clathrin adaptor protein, which in turn redirect HCVne trafficking to the lipid droplets (LDs) via the endosomal-lysosomal pathway. HCVne and LDs co-localization affects the HCV life cycle by enhancing viral replication.

Hepatitis C virus (HCV) infection is associated with hepatic iron overload and elevated serum iron that correlate to poor antiviral responses. Hepcidin (HAMP), a 25-aa cysteine-rich liver-specific peptide, controls iron homeostasis. Its expression is up-regulated in inflammation and iron excess. HCV-mediated hepcidin regulation remains controversial. Chronic HCV patients possess relatively low hepcidin levels; however, elevated HAMP mRNA has been reported in HCV core transgenic mice and HCV replicon-expressing cells. We investigated the effect of HCV core protein on HAMP gene expression and delineated the complex interplay of molecular mechanisms involved. HCV core protein up-regulated HAMP promoter activity, mRNA, and secreted protein levels. Enhanced promoter activity was abolished by co-transfections of core with HAMP promoter constructs containing mutated/deleted BMP and STAT binding sites. Dominant negative constructs, pharmacological inhibitors, and silencing experiments against STAT3 and SMAD4 confirmed the participation of both pathways in HAMP gene regulation by core protein. STAT3 and SMAD4 expression levels were found increased in the presence of HCV core, which orchestrated SMAD4 translocation into the nucleus and STAT3 phosphorylation. To further understand the mechanisms governing the core effect, the role of the JAK/STAT-activating kinase CK2 was investigated. A CK2-dominant negative construct, a CK2-specific inhibitor, and RNAi interference abrogated the core-induced increase on HAMP promoter activity, mRNA, and protein levels, while CK2 acted in synergy with core to significantly enhance HAMP gene expression. Therefore, HCV core up-regulates HAMP gene transcription via a complex signaling network that requires both SMAD/BMP and STAT3 pathways and CK2 involvement.

An estimated 30–40% of patients with chronic hepatitis C have elevated serum iron, transferrin saturation, and ferritin levels. Clinical data suggest that iron is a co-morbidity factor for disease progression following HCV infection. Iron is essential for a number of fundamental metabolic processes in cells and organisms. Mammalian iron homeostasis is tightly regulated and this is maintained through the coordinated action of sensory and regulatory networks that modulate the expression of iron-related proteins at the transcriptional and/or posttranscriptional levels. Disturbances of iron homeostasis have been implicated in infectious disease pathogenesis. Viruses, similarly to other pathogens, can escape recognition by the immune system, but they need iron from their host to grow and spread. Hepcidin is a 25-aa peptide, present in human serum and urine and represents the key peptide hormone, which modulates iron homeostasis in the body. It is synthesized predominantly by hepatocytes and its mature form is released in circulation. In this review, we discuss recent advances in the exciting crosstalk of molecular mechanisms and cell signaling pathways by which iron and hepcidin production influences HCV-induced liver disease.

Polycystin-1 (PC1) has been proposed as a chief mechanosensing molecule implicated in skeletogenesis and bone remodeling. Mechanotransduction via PC1 involves proteolytic cleavage of its cytoplasmic tail (CT) and interaction with intracellular pathways and transcription factors to regulate cell function. Here we demonstrate the interaction of PC1-CT with JAK2/STAT3 signaling axis in mechanically stimulated human osteoblastic cells, leading to transcriptional induction of Runx2 gene, a master regulator of osteoblastic differentiation. Primary osteoblast-like PC1-expressing cells subjected to mechanical-stretching exhibited a PC1-dependent increase of the phosphorylated(p)/active form of JAK2. Specific interaction of PC1-CT with pJAK2 was observed after stretching while pre-treatment of cells with PC1 (anti-IgPKD1) and JAK2 inhibitors abolished JAK2 activation. Consistently, mechanostimulation triggered PC1-mediated phosphorylation and nuclear translocation of STAT3. The nuclear phosphorylated(p)/DNA-binding competent pSTAT3 levels were augmented after stretching followed by elevated DNA-binding activity. Pre-treatment with a STAT3 inhibitor either alone or in combination with anti-IgPKD1 abrogated this effect. Moreover, PC1-mediated mechanostimulation induced elevation of Runx2 mRNA levels. ChIP assays revealed direct regulation of Runx2 promoter activity by STAT3/Runx2 after mechanical-stretching that was PC1-dependent. Our findings show that mechanical load upregulates expression of Runx2 gene via potentiation of PC1-JAK2/STAT3 signaling axis, culminating to possibly control osteoblastic differentiation and ultimately bone formation.

Mechanisms that favor Hepatitis C virus (HCV) persistence over clearance are unclear, but involve defective innate immunity. Chronic infection is characterized by hepatic iron overload, hyperferraemia and hyperferittinaemia. Hepcidin modulates iron egress via ferroportin and its storage in ferritin. Chronic HCV patients have decreased hepcidin, while HCV replication is modified by HAMP silencing. We aimed to investigate interactions between HCV and hepcidin, during acute and chronic disease, and putative alterations in cellular iron homeostasis that enhance HCV propagation and promote viral persistence. Thus, we used HCV JFH-1-infected co-cultures of Huh7.5 hepatoma and THP-1 macrophage cells, HCV patients' sera and Huh7 hepcidin-expressing cells transfected with HCV replicons. Hepcidin levels were elevated in acutely infected patients, but correlated with viral load in chronic patients. HAMP expression was up-regulated early in HCV infection in vitro, with corresponding changes in ferritin and FPN. Hepcidin overexpression enhanced both viral translation and replication. In HCV-infected co-cultures, we observed increased hepcidin, reduced hepatoma ferritin and a concurrent rise in macrophaghic ferritin over time. Altered iron levels complemented amplified replication in hepatoma cells and one replication round in macrophages. Iron-loading of macrophages led to enhancement of hepatic HCV replication through reversed ferritin "flow." Viral transmissibility from infected macrophages to naive hepatoma cells was induced by iron. We propose that HCV control over iron occurs both by intracellular iron sequestration, through hepcidin, and intercellular iron mobilisation via ferritin, as means toward enhanced replication. Persistence could be achieved through HCV-induced changes in macrophagic iron that enhances viral replication in these cells.

// Michalis V. Karamouzis 1 , Georgia Dalagiorgou 1 , Urania Georgopoulou 2 , Afroditi Nonni 3 , Michalis Kontos 4 , Athanasios G. Papavassiliou 1 1 Molecular Oncology Unit, Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece 2 Laboratory of Molecular Virology, Hellenic Pasteur Institute, 11521 Athens, Greece 3 First Department of Pathology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece 4 Department of Propaedeutic Surgery, Medical School, National and Kapodistrian University of Athens, ‘Laikon’ General Hospital, 11527 Athens, Greece Correspondence to: Athanasios G. Papavassiliou, e-mail: papavas@med.uoa.gr Keywords: HER-3, ErbB, dimerization pattern, proximity ligation assay, breast cancer Received: September 13, 2015 Accepted: December 22, 2015 Published: December 26, 2015 ABSTRACT Breast carcinogenesis is a multi-step process in which membrane receptor tyrosine kinases are crucial participants. Lots of research has been done on epidermal growth factor receptor (EGFR) and HER-2 with important clinical results. However, breast cancer patients present intrinsic or acquired resistance to available HER-2-directed therapies, mainly due to HER-3. Using new techniques, such as proximity ligation assay, herein we evaluate the dimerization pattern of HER-3 and the importance of context-dependent dimer formation between HER-3 and other HER protein family members. Additionally, we show that the efficacy of novel HER-3 targeting agents can be better predicted in certain breast cancer patient sub-groups based on the dimerization pattern of HER protein family members. Moreover, this model was also evaluated and reproduced in human paraffin-embedded breast cancer tissues.

The objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model. A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector. In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector. Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity. These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of values as a component of an HSV multi-subunit vaccine.

The type 1 and type 2 glycoprotein G (gGl and gG2) of herpes simplex virus (HSV) were expressed in Escherichia coli as fusion proteins with the maltose binding protein (MBP) using t

International audience; The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99-124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of antit-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection.

In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E. coli as fusion proteins with the MBP or the GST protein. The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays. A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A. Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein. None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens. With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen. The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV.

Aim: Many BC patients present intrinsic or acquired resistance to anti-HER-2 directed therapies, mainly attributed to HER-3. Our goal was to evaluate the dimerization pattern of EGFR, HER2/3/4 with or without the addition of heregulin (Hg) and HER-3 targeting agents. Methods: MCF7 and SKBR3 cell lines were used. Dimerization patterns were studied using proximity ligation assay and immunofluorescence, where proteins in proximity can be visualized by combining classical immunocytochemistry with rolling circle amplification. Concentrations of used reagents were: HRG1-b1 10mM (R&D Systems), Pertuzumab (P) 30mM (Genetech), U3 (U) inhibitor 30mM (U3 Pharma). After blocking, cells were incubated with combination of primary antibodies (antiHER2/HER3, antiHER2/HER4, antiEGFR/HER3, anti-EGFR/HER4) in a preheated humidity chamber for 1 h at 37°C. Cells were then incubated with the PLA probes diluted 1:5 in antibody diluent (Olink Bioscience, Uppsala, Sweden) in a humidified chamber for 1 h at 37°C. Subsequent hybridization, ligation, amplification, and detection were performed as per manufacturer's instruction. Fluorescence images were acquired using a Zeiss Axiovert microscope (Carl Zeiss Microscopy, Thornwood, NY). Data analysis was done using Duolink ImageTool Software that has been developed for quantification of PLA signals. Results: In MCF7 cells (PLA signals scale 0-12), Hg addition increased dimmers EGFR/HER4 (9,7 vs 5,13), EGFR/HER3 (8,07 vs 3,36) and less HER2/HER3 (4,12 vs 3,35). The addition of Hg and P resulted in EGFR/HER4 4,67, EGFR/HER3 2,46, HER2/HER3 3,0 and Hg and U resulted in EGFR/HER4 0,6, EGFR/HER3 0,6, HER2/HER3 0,6. In SKBR3 cells (PLA signals scale 0-60), Hg addition increased dimmers EGFR/HER4 (60 vs 40), HER2/HER3 (26,93 vs 15,92) and less EGFR/HER3 (16 vs 10,6). The addition of Hg and P resulted in EGFR/HER4 17,0, HER2/HER3 19,9, EGFR/HER3 12,5 and Hg and U resulted in EGFR/HER4 0,7, HER2/HER3 0,7, EGFR/HER3 0,7. All images and data analysis will be presented in full detail. Conclusions: The addition of Hg enhances HER3-based dimmer formation, while HER-3 targeting agents lower HER3-containing dimmer formation. The technique can be used in paraffin-embedded BC specimens and may identify patient sub-groups who benefit by HER-3 targeting agents. Disclosure: All authors have declared no conflicts of interest.

In a previous study a novel gene designated as US8.5 was identified in the unique short component of the herpes simplex virus type 1 (HSV1) genome. Epitope tagging experiments suggested the existence of a protein encoded by this gene in HSV1 infected cells. To further analyze the US8.5 gene product and function, a rabbit polyclonal antiserum was raised against a recombinant beta-galactosidase-US8.5 fusion protein expressed in E. coli. This antiserum reacted specifically with a 19 kDa protein in HSV1(F) infected cells as shown by immunoblotting and immunoprecipitation experiments. In addition, using the same antiserum a 21 kDa protein was detected in lysates from cells infected with HSV2(G), which was most likely the HSV2 homolog of US8.5. Kinetic studies indicated that US8.5 is expressed as gamma 1 gene. In addition, the US8.5 protein was immunoprecipitated with the anti-US8.5 serum from 32P-labeled lysates of Vero cells infected with HSV1, demonstrating that the protein is phosphorylated. Immunofluorescence studies localized the US8.5 protein to the nucleoli of HSV1 infected cells.