Jawed Iqbal

Hepatitis C virus activates interleukin-1β via caspase-1-inflammasome complex

Interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine involved in the pathogenesis of HCV, but the sensors and underlying mechanisms that facilitate HCV-induced IL-1β proteolytic activation and secretion remains unclear. In this study, we have identified a signalling pathway leading to IL-1β activation and secretion in response to HCV infection. Previous studies have shown the induction and secretion of IL-1β through the inflammasome complex in macrophages/monocytes. Here, we report for the first time the induction and assembly of the NALP3-inflammasome complex in human hepatoma cells infected with HCV (JFH-1). We demonstrate that activation of IL-1β in HCV-infected cells involves the proteolytic processing of pro-caspase-1 into mature caspase-1 in a multiprotein inflammasome complex. Next, we demonstrate that HCV is sensed by NALP3 protein, which recruits the adaptor protein ASC for the assembly of the inflammasome complex. Using a small interfering RNA approach, we further show that components of the inflammasome complex are involved in the activation of IL-1β in HCV-infected cells. Our study also demonstrates the role of reactive oxygen species in HCV-induced IL-1β secretion. Collectively, these observations provide an insight into the mechanism of IL-1β processing and secretion, which is likely to provide novel strategies for targeting the viral or cellular determinants to arrest the progression of liver disease associated with chronic HCV infection.

Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses

The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-β production during herpesvirus (KSHV, EBV, HSV-1) infections.

BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses

The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

Mechanism of Hepatitis C Virus (HCV)-induced Osteopontin and Its Role in Epithelial to Mesenchymal Transition of Hepatocytes

Background: The mechanism underlying HCV-induced OPN remains elusive. Results: HCV-induced Ca2+ signaling, oxidative stress, and activation of AP-1 and Sp1 play a critical role in OPN activation. Conclusion: OPN plays important roles in EMT and cell migration induced by HCV through the activation of Akt, GSK-3β, and β-catenin. Significance: Our findings provide a novel implication of OPN in HCV-induced HCC. Osteopontin (OPN) is a secreted phosphoprotein, originally characterized in malignant-transformed epithelial cells. OPN is associated with tumor metastasis of several tumors and is overexpressed in hepatocellular carcinoma (HCC) tissue involving HCC invasion and metastasis. Importantly, OPN is significantly up-regulated in liver injury, inflammation, and hepatitis C virus (HCV)-associated HCC. However, the underlying mechanisms of OPN activation and its role in HCV-mediated liver disease pathogenesis are not known. In this study, we investigated the mechanism of OPN activation in HCV-infected cells. We demonstrate that HCV-mediated Ca2+ signaling, elevation of reactive oxygen species, and activation of cellular kinases such as p38 MAPK, JNK, PI3K, and MEK1/2 are involved in OPN activation. Incubation of HCV-infected cells with the inhibitors of AP-1 and Sp1 and site-directed mutagenesis of AP-1- and Sp1-binding sites on the OPN promoter suggest the critical role of AP-1 and Sp1 in OPN promoter activation. In addition, we show the in vivo interactions of AP-1 and Sp1 with the OPN promoter using chromatin immunoprecipitation assay. We also show the calpain-mediated processing of precursor OPN (∼75 kDa) into ∼55-, ∼42-, and ∼36-kDa forms of OPN in HCV-infected cells. Furthermore, we demonstrate the critical role of HCV-induced OPN in increased phosphorylation of Akt and GSK-3β followed by the activation of β-catenin, which can lead to EMT of hepatocytes. Taken together, these studies provide an insight into the mechanisms of OPN activation that is relevant to the metastasis of HCV-associated HCC.

The hepatitis C virus-induced NLRP3 inflammasome activates the sterol regulatory element-binding protein (SREBP) and regulates lipid metabolism

Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with chronic HCV.

Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.

Nuclear Innate Immune DNA Sensor IFI16 is Degraded During Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency

ABSTRACT IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposis sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation. IMPORTANCE Like all herpesviruses, latency is an integral part of the life cycle of Kaposis sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.

Hepatitis C virus nonstructural protein 5A favors upregulation of gluconeogenic and lipogenic gene expression leading towards insulin resistance: a metabolic syndrome

Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.

ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its natural in vivo target cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na+/H+ exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. IMPORTANCE Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.

Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis.

AIM To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus (HCV) nonstructural protein 5A (NS5A). METHODS The human hepatoma cell lines, Huh7 and Huh7.5, were infected with HCV or transiently-transfected with a vector expressing HCV NS5A. The effect of HCV NS5A on the status of the critical players involved in insulin signaling was analyzed using real-time quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0. RESULTS To investigate the effect of insulin treatment on the players involved in insulin signaling pathway, we analyzed the status of insulin receptor substrate-1 (IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1 (Ser(307)) in HCV infected or NS5A transfected Huh7.5 cells compared to their respective controls. Furthermore, an increased phosphorylation of Akt (Ser(473)) was observed in HCV infected and NS5A transfected cells compared to their mock infected cells. In contrast, we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue (Ser(307)) followed by decreased phosphorylation of Akt Thr(308), Fox01 Ser(256) and GSK3β Ser(9), the downstream players of the insulin signaling pathway. Furthermore, increased expression of PECK and glucose-6-phosphatase, the molecules involved in gluconeogenesis, in HCV NS5A transfected cells was observed. CONCLUSION Taken together, our results suggest the role of HCV NS5A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.