Virus/STAT3 interactions: Summary of observations and employed methods.

<p>Virus/STAT3 interactions: Summary of observations and employed methods.</p

Regulatory circuits of the STAT3 signaling pathway.

<p>STAT3 can be activated by a wide range of ligands binding to cytokine, growth factor, or G-protein-coupled receptors. With the exception of receptor tyrosine kinases, these receptors lack intrinsic kinase activity and thus act by recruiting adaptor kinases (e.g., JAKs, SRC) to propagate downstream signals. As a result, STAT3 is phosphorylated at tyrosine 705 (pY705, pink), forms homodimers or heterodimers, and translocates to the nucleus, where it transcribes regulators of various cellular processes. Additionally, STAT3 can be phosphorylated at serine 727 (pS727, purple) by serine/threonine kinases (e.g., MAPK, mTOR, PKCδ), which enhance STAT3 transcriptional activity in the nucleus or direct STAT3 to mitochondria. Acetylation at lysine 685 (K685, red) by histone acetyltransferases (e.g., CREB binding protein CBP/histone acetyltransferase p300) or methylation at lysine 140 (K140, blue) by histone methyltransferases (e.g., SET9) favor or impair STAT3 transcriptional activity, respectively. Unphosphorylated STAT3 exhibits regulatory functions in the nucleus or can be retained in the cytoplasm, where it associates with microtubules and focal adhesions. The activity of STAT3 is tightly regulated by phosphatases (e.g., PTPRD), SOCS3, PIAS3, and miRNAs that fine-tune the temporal pattern of STAT3 activity and its other pathway components. All miRNAs are degrading the mRNAs of the indicated proteins. A, acetylation; CBP, CREB-binding protein; CT-1R, cardiotrophin 1 receptor; CNTFR, ciliary neurotrophic factor receptor; DUSP2, dual specificity protein phosphatase 2; EGFR, epidermal growth factor receptor; GHR, growth hormone receptor; G-CSFR, granulocyte colony-stimulating factor receptor; GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor; gp130, glycoprotein 130; IFNAR, interferon alpha receptor; IFNGR, interferon gamma receptor; IL, interleukin; JAK, Janus kinase; K140, lysine 140; K685, lysine 685; LIFR, leukemia inhibitory factor receptor; MAPK, mitogen-activated protein kinase; M, methylation; miRNA, microRNA; mTOR, mechanistic target of rapamycin; OSMR, oncostatin-M-specific receptor; P, phosphorylation; p300, histone acetyltransferase p300; PDGFR, platelet-derived growth factor receptor; PIAS3, protein inhibitor of activated STAT protein 3; PKCδ, protein kinase C delta type; pS727, phospho-serine 727; PTPRC, receptor-type tyrosine-protein phosphatase C; PTPRD, receptor-type tyrosine-protein phosphatase D; PTPRT, receptor-type tyrosine-protein phosphatase T; pY705, phospho-tyrosine 705; SET9, histone-lysine N-methyltransferase SET9; SOCS3, suppressor of cytokine signaling 3; SRC, proto-oncogene tyrosine-protein kinase; STAT3, signal transducer and activator of transcription 3; TpoR, thrombopoietin receptor; TRIM28, tripartite motif-containing protein 28.</p

Viral replicative advantages and pathological consequences related to STAT3-altered function.

<p><b>(A)</b> Virus-induced perturbation of STAT3 as regulator of apoptosis. In the context of viral infections, apoptosis can be restrained via STAT3, since it favors the expression of antiapoptotic factors (e.g., <i>PCBP2</i> and <i>BIRC5</i>) or prevents proapoptotic ones (e.g., RTA, <i>FOS</i>, <i>JUN</i>, and <i>NR4A2</i>). In contrast, inhibition of STAT3 by viruses such as IAV and MuV has been associated with the induction of the apoptotic process. <b>(B)</b> Viral manipulation of STAT3 and its effect on immune responses. Viral inhibition of STAT3 can induce a decrease of ISG and APR gene expression and favor immune evasion, as in the case of KSHV and HEV. Virus-mediated STAT3 activation can also have immunosuppressive actions such as impairing DC function (KSHV and HCMV) and favoring the expansion of MDSCs (HCV). In other cases, the proinflammatory actions of STAT3 have been associated with the development of host pathologies such as cancer (KSHV). <b>(C)</b> Virus-induced alteration of STAT3 and its impact on cell and tissue organization. STAT3 activation during HCV infection has been associated with alterations of the MT network. This represents a potential advantage for HCV by favoring virus trafficking along MTs. At the tissue and organ level, STAT3 activation has been associated with the development of fibrosis (HCV), the disruption of endothelial vascular junctions (IAV), and enhanced cell invasion, which favors cancer development (EBV). ANGPTL4, angiopoietin-like protein 4; APR, acute phase response; BIRC5, baculoviral IAP repeat-containing protein 5; CCL5, C-C motif chemokine ligand 5; DCs, dendritic cells; DC-SIGN, dendritic cell-specific ICAM-3-grabbing non-integrin 1; EBV, Epstein–Barr virus; FOS, proto-oncogene c-Fos; HCC, hepatocellular carcinoma; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HEV, hepatitis E virus; HSCs, hepatic stellate cells; IAV, influenza A virus; IDO1, indoleamine 2,3-dioxygenase 1; IL-10, interleukin 10; ISG, interferon-stimulated gene; JUN, proto-oncogene c-Jun; KSHV, Kaposi’s sarcoma-associated herpesvirus; MCL1, induced myeloid leukemia cell differentiation protein Mcl-1; MDSCs, myeloid-derived suppressor cells; MT, microtubule; MUC1, mucin 1 cell surface associated; MuV, mumps virus; NPC, nasopharyngeal carcinoma; NR4A2, nuclear receptor subfamily 4 group A member 2; PCBP2, poly(rC)-binding protein 2; PD-L1, programmed cell death 1 ligand 1; RTA, R transactivator; RVFV, Rift Valley fever virus; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor beta; T_reg, regulatory CD4⁺ T cell; VZV, varicella-zoster virus.</p

Ectopic expression of apoE dose-dependently stimulates HCV production.

<p>(A) Schematic of apoE mutants and apoE-derived peptide sequence. Receptor binding domain (RBD: amino acids 136–150) and heparan sulfate proteoglycan binding domain (HSPG-BD: amino acids 142–147) are represented. Mutations of the apoE HSPG-BD (apoEΔHSPG-BD, apoE K143A, K146A, and apoE R142A, R145A) were generated by site-directed mutagenesis. (B) Huh7.5.1 cells were either co-electroporated (Co-EP) with luciferase-encoding HCV RNA (Luc-Jc1) and siRNA targeting endogenous apoE expression (siApoE) (2–7) or mock-transfected (1). 24 h post-transfection, cells were transduced with adenoviruses expressing GFP (Ad-CTRL) as a control, or with increasing concentrations of adenoviruses expressing wt apoE (Ad-apoE-wt), representing 1∶100–1∶5 dilutions, and numbered from 2 to 7 according to increasing concentration. Three days post-transduction, intracellular apoE, actin and HCV core expression was determined by immunoblot of cell lysates. (C) Extracellular culture supernatants of the cells from (B) with corresponding number designations were concentrated by sucrose cushion. ApoE, HCV E2, and core expression were tested by Western blot. (D) HCV infection from apoE modulated cells was conducted by exposing naïve Huh7.5.1 cells to culture media from cells transfected with HCV RNA and transduced with increasing concentrations of Ad-apoE-wt or with Ad-CTRL with number designations corresponding to (B) and (D). 3d post-infection, infectivity was measured by luciferase reporter activity. HCVcc infection is expressed as a percentage relative to apoE-silenced cells transduced with Ad-CTRL. Results are expressed as mean±SD of the experiment performed in triplicate (** = <i>P</i><0.001).</p

Viral manipulation of the STAT3 signaling pathway.

<p><b>(A)</b> Viruses activating STAT3 function and the mechanisms involved. Viral proteins such as HBx, NS5A, core, NSs, EBNA2, LMP1, US28, and IE1 induce STAT3 activation either directly or by favoring the action of upstream positive regulators. Viruses like HCMV and KSHV code for homologues of human interleukins such as IL-10 and IL-6. Alternatively, virus-induced activation of STAT3 can be achieved by the inhibition of negative regulators such as SOCS3, PTPRD, TRIM28, and Let-7a. In the case of some viruses, STAT3 activation (VZV and ZIKV) or STAT3-mediated effects (IAV) have been described, but the mechanisms involved have not been fully elucidated. All miRNAs are degrading the mRNAs of the indicated proteins. <b>(B)</b> Viruses suppressing STAT3 function and the mechanisms involved. Virus-mediated inactivation of STAT3 can be attained by decreasing its phosphorylation (KSHV, IAV, and hMPV), inducing STAT3 protein degradation (MuV), hampering its transcriptional activity (MeV), or altering its subcellular localization (HCMV, RABV, HEV, and hMPV). EBNA2, Epstein–Barr virus nuclear antigen 2; EBV, Epstein–Barr virus; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HEV, hepatitis E virus; hMPV, human metapneumovirus; IAV, influenza A virus; IE1, intermediate-early protein 1; IL-6, interleukin 6; IL-10, interleukin 10; IRAK1, interleukin 1 receptor-associated kinase 1; JAK1, Janus kinase 1; KSHV, Kaposi’s sarcoma-associated herpesvirus; LMP1, latent membrane protein 1; miRNA, microRNA; MeV, measles virus; MK2, mitogen-activated protein kinase 2; MuV, mumps virus; NS5A, non-structural protein 5A; NSs, non-structural proteins; P, phosphorylation; PKCδ, protein kinase C delta type; PTPRD, receptor-type tyrosine-protein phosphatase D; RABV, rabies virus; ROS, reactive oxygen species; RVFV, Rift Valley fever virus; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; TRIM28, tripartite motif-containing protein 28; u-STAT3, unphosphorylated STAT3; vIL-10, viral IL-10; vIL-6, viral IL-6; VZV, varicella-zoster virus; ZIKV, Zika virus.</p

Syndecan 4 is involved in HCV infection.

<p>(A) Huh 7.5.1 cells were transfected with oligonucleotides to knockdown expression of SDC1 (siSDC1), SDC4 (siSDC4), CD81 (siCD81) or a siRNA control (siCTRL). Three days post-transfection, the transfected-cells were analyzed for SDC1 (black bars) and SDC4 (gray bars) mRNA expression. (B) HCVcc infection was quantified in syndecan-modulated cells by assaying HCV RNA levels by qRT-PCR 3d after exposing cells to Jc1 HCVcc for 4 h at 37°C (MOI = 1) followed by three washes and replacement of media (* = <i>P</i><0.01, ** = <i>P</i><0.001). (C) Huh 7.5.1 cells were transfected with oligonucleotides to knockdown expression of SDC1, SDC4 using a smartpool containing 4 siRNA (siSDC4), SDC4 using siRNA aliquoted individually from siSDC4 (siSDC4-1 to siSDC4-4), CD81 (siCD81) or a siRNA control (siCTRL). Three days post-transfection, transfected cells were infected with Luc-Jc1 HCVcc for 4 h at 37°C. Three days post-infection, infectivity was measured by luciferase reporter activity. HCVcc infection is expressed as a percentage relative to siCTRL-silenced cells (* = <i>P</i><0.01, ** = <i>P</i><0.001). (D) Huh 7.5.1 cells were transfected with oligonucleotides to knockdown expression of SDC4 (siSDC4) or a siRNA control (siCTRL). 24 h post-transfection, cells were transduced with adenoviruses expressing either GFP (Ad-CTRL) as a control, Ad-HA-SDC4-wt or Ad-HA-SDC4-Y180L. Three days post-transduction, transfected and transduced cells were infected with Luc-Jc1 HCVcc for 4 h at 37°C. Three days post-infection, infectivity was measured by luciferase reporter activity. HCVcc infection is expressed as a percentage relative to siCTRL-silenced cells transduced with Ad-CTRL (* = <i>P</i><0.01). (E) HCV pseudoparticle (HCVpp) entry was analyzed by exposure of naïve Huh 7.5.1 cells to sucrose-cushion purified HCVpp (J6) at a MOI of 1 for 4 h at 37°C. Two days after, HCVpp entry was quantified by measuring luciferase activity. Bars represent means±SD of three independent experiments performed in triplicate, of percent change relative to siCTRL. (F) Very-low density lipoprotein (VLDL) at concentrations of 0.5, 5, and 50 µg/mL or PBS as a control were co-incubated with Luc-Jc1 HCVcc (MOI = 1) with naïve Huh 7.5.1 cells for 2 h at 4°C. Following incubation, cells were washed three times with PBS and viral RNA attachment was assessed by qRT-PCR. Bars represent means±SD of three independent experiments performed in triplicate. (* = <i>P</i><0.01, ** = <i>P</i><0.001).</p

Ectopic expression of apoE dose-dependently stimulates HCV production.

<p>(A) Schematic of apoE mutants and apoE-derived peptide sequence. Receptor binding domain (RBD: amino acids 136–150) and heparan sulfate proteoglycan binding domain (HSPG-BD: amino acids 142–147) are represented. Mutations of the apoE HSPG-BD (apoEΔHSPG-BD, apoE K143A, K146A, and apoE R142A, R145A) were generated by site-directed mutagenesis. (B) Huh7.5.1 cells were either co-electroporated (Co-EP) with luciferase-encoding HCV RNA (Luc-Jc1) and siRNA targeting endogenous apoE expression (siApoE) (2–7) or mock-transfected (1). 24 h post-transfection, cells were transduced with adenoviruses expressing GFP (Ad-CTRL) as a control, or with increasing concentrations of adenoviruses expressing wt apoE (Ad-apoE-wt), representing 1∶100–1∶5 dilutions, and numbered from 2 to 7 according to increasing concentration. Three days post-transduction, intracellular apoE, actin and HCV core expression was determined by immunoblot of cell lysates. (C) Extracellular culture supernatants of the cells from (B) with corresponding number designations were concentrated by sucrose cushion. ApoE, HCV E2, and core expression were tested by Western blot. (D) HCV infection from apoE modulated cells was conducted by exposing naïve Huh7.5.1 cells to culture media from cells transfected with HCV RNA and transduced with increasing concentrations of Ad-apoE-wt or with Ad-CTRL with number designations corresponding to (B) and (D). 3d post-infection, infectivity was measured by luciferase reporter activity. HCVcc infection is expressed as a percentage relative to apoE-silenced cells transduced with Ad-CTRL. Results are expressed as mean±SD of the experiment performed in triplicate (** = <i>P</i><0.001).</p

Anti-CD81 mAb inhibits HCV cell-to-cell transmission and viral spread.

<p>(A) Quantification of HCV-infected target cells (Ti) after co–cultivation with HCV producer cells (Pi) during incubation with control or anti-CD81 QV-6A8-F2-C4 mAbs (10 µg/ml) in the presence of neutralizing anti-HCV E2 mAb (AP33, 25 µg/ml) by flow cytometry. (B) Percentage of infected target cells is shown as histograms and is represented as means ± SD from three experiments. (C) Long-term analysis of HCVcc infection in the presence or absence of control or anti-CD81 QV-6A8-F2-C4 mAbs at the indicated concentrations. Antibodies were added 48 h after HCVcc infection and control medium or medium containing mAbs were replenished every 4 days. Luciferase activity was determined in cell lysates every 2 days. Data are expressed as Log₁₀ RLU and represent means ± SD of three experiments performed in duplicate. (D) Cell viability after long-term exposure to anti-CD81 mAb QV-6A8-F2-C4. Cell viability was assessed using MTT assay after incubation of Huh7.5.1 cells for 14 days in the presence or absence of control or anti-CD81 mAbs at 1, 10, or 100 µg/ml. Data are expressed as % cell viability relative to cells incubated in the absence of mAb and represent means ± SD from one experiment performed in triplicate. (E–F) Virus spread in the presence or absence of anti-CD81 mAbs QV-6A8-F2-C4 (E) and JS81 (F). Antibodies (50 µg/ml) were added 48 h after HCVcc (Jc1) infection and control medium or medium containing antibodies were replenished every 4 days. HCV-infected cells were visualized 9 days post-infection via immunofluorescence using anti-NS5A (E) or anti-E2 (CBH23) (F) antibodies. The percentage of infected cells was calculated as the number of infected cells relative to the total number of cells as assessed by 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei.</p

Synergistic effect of anti-envelope and anti-CD81 antibodies on inhibition of HCV infection.

<p>HCVpp of strains P02VJ or HCVcc-Luc Jc1 were pre-incubated with anti-E2 mAb IGH461 or purified heterologous anti-HCV IgG (1 or 10 µg/ml) obtained from an unrelated chronically infected subject or isotype control IgG for 1 hour at 37°C and added to Huh7.5.1 cells pre-incubated with serial dilutions of anti-CD81 QV-6A8-F2-C4 or rat isotype control mAbs. HCVpp entry and HCVcc infection were analyzed by luciferase assay. The Combination Index (CI) was calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#pone.0064221-Zhu1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#pone.0064221-Zhao1" target="_blank">[43]</a>. A CI less than 0.9, between 0.9 and 1.1, and more than 1.1 indicates synergy, additivity, and antagonism, respectively. CI for anti-CD81 mAb in combination with 10 µg/ml anti-HCV IgG in HCVpp entry inhibition was calculated for an IC₇₅ as the combination resulted in an inhibition below the IC₅₀ and is indicated by a star (*). IC₅₀ of anti-envelope antibodies: anti-E2, 70±5 µg/ml (for HCVpp); anti-HCV IgG, 40±3 µg/ml (for HCVpp), 120±6 µg/ml (for HCVcc).</p

Anti-CD81 mAbs dose-dependently inhibit HCV infection.

<p>(A–B) Dose-dependent inhibition of HCV infection by anti-CD81 mAbs. Huh7.5.1 cells were pre-incubated with increasing concentrations of anti-CD81 or isotype control (CTRL IgG) mAbs for 1 h at 37°C before infection with (A) HCVcc (Luc-Jc1 (2a)) or (B) HCVpp (HCV-J (1b)). Three days later, viral infection was quantitated by assessing the expression of luciferase reporter gene. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064221#s3" target="_blank">Results</a> are expressed as % HCVcc infection or % HCVpp entry and represent means ± SD of one representative experiment performed in triplicate. (C) Inhibition of infection of HCVpp bearing envelope glycoproteins from genotypes 1–6. Huh7.5.1 cells were pre-incubated with a fixed concentration (100 µg/ml) of antibodies before infection with HCVpp (strains H77 (1a), JFH1 (2a), UKN3A1.28 (3a), UKN4.21.16 (4), UKN5.14.4 (5), UKN6.5.340 (6)). Means ± SD from a representative experiment performed in triplicate are shown.</p