Fahed Parvaiz

Hepatitis C Virus Infection: Molecular Pathways to Insulin resistance

Chronic Hepatitis C virus has the potential of inducing insulin resistance and type 2 Diabetes Mellitus in vitro as well as in vivo . Structural and non-structural proteins of HCV modulate cellular gene expression in such a way that insulin signaling is hampered, concomitantly leads toward diabetes mellitus. A number of mechanisms have been proposed in regard to the HCV induced insulin resistance involving the upregulation of Inflammatory cytokine TNF-α, hypophosphorylation of IRS-1 and IRS-2, phosphorylation of Akt, up-regulation of gluconeogenic genes, accumulation of lipids and targeting lipid storage organelles. This review provides an insight of molecular mechanisms by which HCV structural and non-structural proteins can induce insulin resistance.

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.

Current and future therapies for hepatitis C virus infection: from viral proteins to host targets

Hepatitis C virus (HCV) infection is the most important problem across the world. It causes acute and chronic liver infection. Different approaches are in use to inhibit HCV infection, including small organic compounds, siRNA, shRNA and peptide inhibitors. This review article summarizes the current and future therapies for HCV infection. PubMed and Google Scholar were searched for articles published in English to give an insight into the current inhibitors against this life-threatening virus. HCV NS3/4A protease inhibitors and nucleoside/nucleotide inhibitors of NS5B polymerase are presently in the most progressive stage of clinical development, but they are linked with the development of resistance and viral breakthrough. Boceprevir and telaprevir are the two most important protease inhibitors that have been approved recently for the treatment of HCV infection. These two drugs are now the part of standard-of-care treatment (SOC). There are also many other drugs in phase III of clinical development. When exploring the various host-cell-targeting compounds, the most hopeful results have been demonstrated by cyclophilin inhibitors. The current SOC treatment of HCV infection is Peg-interferon, ribavirin and protease inhibitors (boceprevir or telaprevir). The future treatment of this life-threatening disease must involve combinations of therapies hitting multiple targets of HCV and host factors. It is strongly expected that the near future, treatment of HCV infection will be a combination of direct-acting agents (DAA) without the involvement of interferon to eliminate its side effects.

An overview: In vitro models of HCV replication in different cell cultures

Although much of productive research has been conducted in the field of molecular virology of Hepatitis C virus (HCV) regarding its genes, gene functions and proteins, development of an efficient cell culture model for its replication remained a focused area. Focus has been directed to establish HCV in vitro replication system. This replication system should mimic its intrahepatic pathogenesis so that antivirals should be screened and in vitro gene profiling of HCV induced pathogenesis should be worked out. Since 1990 various experimental approaches and strategies have been utilized in phase of development of a robust replication model for HCV, and success has been reported for a few genotypes. Still the work is going on to have more success in availing such robust replication models for all the genotypes. This will help to have a common antiviral strategy against HCV induced pathogenesis involving any genotype or subtype.

Predictive Potential of IL-18 −607 and Osteopontin −442 Polymorphism in Interferon-Based Therapy of HCV Infection in the Pakistani Population

The adaptive immune system plays an important role in response to interferon plus ribavirin treatment of hepatitis C virus (HCV) infection. Cytokines play a significant role in the adaptive immune system. The production of cytokines may be regulated by single nucleotide polymorphisms (SNPs). This study was designed to examine the correlation of some important SNPs of cytokines with interferon plus ribavirin treatment of HCV infection in the Pakistani population. We followed 140 chronic HCV-infected patients in our study. All of these patients had completed their planned course of interferon plus ribavirin treatment. We also considered 120 healthy subjects as controls. The detection of interleukin-18 (IL-18) SNPs was performed by tetra-primers amplification-refectory mutation system polymerase chain reaction, while for genotyping of osteopontin (OPN), transforming growth factor beta (TGFβ), and N-acetylgalactosaminyltransferase 8 (GALNT8) SNPs, allele-specific polymerase chain reaction was performed. The distribution of the IL-18 -607AA genotype varied significantly between healthy control and patient groups. Its distribution was significantly high in healthy subjects than HCV patients (p = 0.031), signifying its potential involvement in the natural clearance of HCV infection. The occurrence of the -607AA genotype of IL-18 was also significantly higher in the sustained virological group (SVR) than in the nonresponder (NR) group (p = 0.046), highlighting its protective involvement in the treatment outcome of chronic HCV infection. The frequency of the OPN -442TT genotype was higher in the SVR group than in the NR group (p = 0.034), indicating a significant possible role of this genotype in therapy for HCV infection. No important association was found between TGFβ and GALNT8 genotypes and the natural clearance and treatment response of HCV infection. IL-18 -607AA and OPN -442TT genotypes can be used as positive predictive markers of interferon plus ribavirin treatment of HCV infection in the Pakistani population.

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.

Ionotropic Purinergic Receptors P2X4 and P2X7: Proviral or Antiviral? An Insight into P2X Receptor Signaling and Hepatitis C Virus Infection

Purinergic P2X receptors are plasma membrane bound, ATP-gated ion channels that are expressed on wide range of cells and respond to varying ATP concentrations in extracellular environment. Upon activation they increase membrane permeability for Ca(2+) ions and trigger a cascade of signaling complexes. During the course of hepatitis C virus (HCV) infection, ATP is released from the infected hepatocyte, which binds with Purinergic receptors (P2X) on peripheral blood mononuclear cells (PBMCs) and initiate downstream signaling pathways by disturbing the ionic balance of the cell. The present study investigates quantitative expression of P2X7 and P2X4 along with selected host genes PEPCK, transforming growth factor β (TGF-β), MAPK, Rho, and Akt in PBMCs of chronic HCV infection patients. PBMCs were isolated from collected blood samples of study subjects. Transcript analysis of P2X7, P2X4, and targeted downstream genes was done using quantitative real-time polymerase chain reaction. Relative expression analysis was performed by unpaired Students t test on GraphPad Prism version 5. We found a notable increase of threefolds and 1.8-folds in the expression of P2X7 and P2X4 receptors in treatment naïve category while the expression of PEPCK, TGF-β, MAPK, AKT, and Rho A increased by 2.8, 1.9, 2.2, 2.2, and 1.8-folds, respectively. In sustained virological response patients, P2X7 significantly increased up to 3.5-folds while the expression of P2X4 receptor was increased up to twofold. In third category, treatment nonresponder, the expression of P2X7, P2X4 receptors, and targeted markers remained un-altered. This study deals with two major aspects of P2X4 and P2X7 receptors in PBMCs of chronic HCV individuals. One is their role in providing antiviral immunity to host against HCV; second aspect is the role of P2X receptors in inducing HCV pathogenesis via AKT, TGF-β, Rho A, PEPCK, and MAPK expression.

Quantitative evaluation of insulin resistance markers in Pakistani patients suffering from HCV-associated type 2 diabetes mellitus

The current study was designed to determine the role of the host genes involved in the development of chronic hepatitis C-associated type 2 diabetes mellitus. This study was carried out in patients in four different stages of chronic hepatitis C virus (HCV) infection, including treatment-naïve HCV patients, HCV-positive patients with type 2 diabetes mellitus (T2DM), non-responders and responders. The mRNA expression level of host genes, such as glucose-6-phosphatase (G6Pase), tumor necrosis factor α (TNF-α) and different adipokines including adiponectin, leptin and resistin, was quantified and compared to healthy controls. HCV infection was found to be associated with insulin resistance, a step towards type 2 diabetes mellitus (T2DM). The results also suggest the potential role of adipokines in chronic HCV (CHC)-associated T2DM. The upregulation of gluconeogenic genes, such as G6Pase and resistin, and a decreased mRNA expression level of adiponectin suggest the potential role of selected markers in the CHC-associated T2DM in Pakistani population. Based on these results, it is concluded that upregulation of TNF-α, G6Pase and resistin in chronic HCV patients leads to gluconeogenesis, eventually favoring T2DM. Collectively, these findings suggest that CHC patients are more prone to T2DM.

In silico analysis of Glycinamide ribonucleotide transformylase inhibition by PY873, PY899 and DIA

In humans, purine de novo synthesis pathway consists of multi-functional enzymes. Nucleotide metabolism enzymes are potential drug targets for treating cancer and autoimmune diseases. Glycinamide ribonucleotide transformylase (GART) is one of the most important trifunctional enzymes involved in purine synthesis. Previous studies have demonstrated the role of folate inhibitors against tumor activity. In this present study, three components of GART enzyme were targeted as receptor dataset and in silico analysis was carried out with folate ligand dataset. To accomplish the task, Autodock 4.2 was used for determining the docking compatibilities of ligand and receptor dataset. Taken together, it has been suggested that folate ligands could be potentially used as inhibitors of GART.

In-Vitro Transcription analysis of NS5A from HCV-3a circulating in Pakistani patients with chronic hepatitis C and their differential response to antiviral therapy

Objective: Mutations in HCV nonstructural protein 5A (NS5A) play a vital role in virus resistance. The aim of this study was to develop a correlation between NS5A mutations (genotype 3a) and virological response towards interferon alpha (IFN-α) plus ribavirin therapy. Methods: In this study, which was conducted from 09-02-2013 to 25-11-2015 in the rural area of Province Sindh – Pakistan, total patients’ responses to peg-IFN therapy were investigated. Patients were given peg-IFN therapy for 24 to 48 weeks and categorized as sustained virologic responders (SVR) or non-responders (NR) to HCV infection. HCV NS5A region (2215-2335) of genotype 3a was identified in both responders and non-responders. Results: Twenty-four NR with 24 SVR isolates showed significant mutations within the nonstructural protein 5A region in HCV genotype 3a. The New Zealand (NZL1) (GenBank D17763) differences were observed by using gene. The ISDR mutations for nonstructural protein 5A in non-responders have been reported as a possible explanation of HCV interferon resistance. Conclusion: Based on these results, it is suggested that decreased SVR is caused by the increased mutations in nonstructural protein 5A sequences. When the sequence outside the Protein kinases R binding domain (PKRBD) (2281–2335) was examined, significant differentiations were observed among the SVR and NR classes at few amino acid strains.