Study of HCV Induced IL-12 Expression by Inhibition of JNK Activated NF-kB and AP-1 Pathways

Abstract

Hepatitis C virus (HCV) has infected more than 200 million patients worldwide. It triggers coordinated innate and adaptive responses in host cells. HCV genome and proteins of the replicating virus are recognized as non-self-antigens by host cells to activate Toll Like Receptors (TLRs). Activated TLRs ultimately express cytokines, which can clear virus either by activating interferon (IFN), protein kinase C (PKC) and RNAse L system or through activation of cytotoxic T-lymphocytes. Interleukin-12 (IL-12) is a one of the antiviral cytokine, capable of clearing HCV by bridging both innate and adaptive antiviral immune responses. Activation of TLR-4 on macrophages surface induces expression of IL-12 via NF-κB and AP- 1 transcriptional pathways. After expression, IL-12 releases IFN γ, which activates anti-HCV cytotoxic lymphocytes. Conversely, in chronic HCV infection down-regulation of IL-12 has been reported by a number of studies. This study was designed to evaluate HCV-core mediated down-regulation of IL-12 transcriptional pathway by employing a logical modeling approach based on the Ren´e Thomas formalism and insilico Protein-Protein interaction (PPI) studies. The Biological regulatory network (BRN) was consisted of TLR-4, NF-κB, IL-12, IFN- γ and HCV-Core protein. In Thomas formalism activation of a protein in BRN is denoted “1” and inhibition with “0”. The logical parameters of entities were estimated by using SMBioNet. The differential expression of IL-12p70 and IL-12p40 was measured in Normal (n=20), Acute HCV infection (n=20), Responders (n=20) and Non-responders (n=20) (NR) by ELISA. The PPIs were performed by HADDOCK and ZDOCK webservers. Results demonstrated a steady state of [1, 0, 0, 0, 1] which was designated chronic stage of infection i.e. though TLR-4 triggered the immune response but HCV adopt immune evasion and lead to chronic infection. HCV also adopted different trajectories to accomplish the persistence of chronic phase of infection. It also implicated that human immune system tries to clear HCV but HCV Core protein is capable of inducing system oscillations to evade the immunity. The expression of the IL-12p70 was comparatively high in acute phase patients (116.85 pg/ml) as compared to chronic (52 pg/ml), and non-responders (32 mg/ml) Responders (63.68 pg/ml). Similarly, IL-12p40 was 179 pg/ml in acute phase infections and NRs having concentration of 98 pg/ml while normal, chronic and responders were 111 pg/ml, 107 pg/ml and 170 pg/ml respectively. IL-12 expression is down regulated by interacting Arg117, Glu89, Tyr81 and Arg149 core residues with NF-kB. While, amino acid residues of core protein involved in hydrogen bonding are Tyr81, Leu185, Arg40, Arg 156 with Ser267, Cys367, Ala318, Leu266, Tyr271 and Thr317 of AP-1. These interactions will be the target of future antiviral therapies. In the second part of the study HCV core gene was synthesized to investigate its interaction with IL-12 expression pathways. Synthetic biology provides tools to design “for the purpose” building of genome/gene and organisms. Approaches have been adopted for the construction of different viral genomes like polio, etc. Annealing temperature difference oligo’s and error prone amplification of gene is the main hurdle in gene synthesis. Results suggested that annealing temperature difference could be optimized by use of the crowding agents. The error may include insertion, deletion, substitution. These errors/mutations mainly arise from incorrect chemical synthesis of oligo’s or polymerase induced errors. Therefore, a sophisticated/efficient error correction method can give better output of desired function. In future we will synthesize full length HCV replicon of different genotypes and will observe their interaction with host cellular proteins.