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Research of Immune Activation by HCV Via TLR7 and Function Analyse for TLR7 Cytoplasmic Domain

Author: WeiWei
Tutor: SunShuHan
School: Second Military Medical University
Course: Genetics
Keywords: HCV Toll-like receptor TIR domain NF-κB MyD88
CLC: R512.63
Type: Master's thesis
Year: 2011
Downloads: 110
Quote: 1
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Abstract

HCV is a positive single-stranded RNA virus able to infect host cells, triggering the body of viral hepatitis, liver fibrosis and even liver cancer occurrence. HCV infection is often accompanied by chronic inflammation caused by the body's innate immune response, many pattern recognition receptors involved in this process. Toll-like receptors are a class of an important class of pattern recognition receptors by identify pathogen associated molecular patterns, the body's natural immune mediated, and can serve as a bridge to connect the body's natural immunity and acquired immunity. Toll-like receptor able to recognize the inherent component of a variety of pathogenic microorganisms, mediated by the immune response of the body against a variety of pathogenic bacteria and viruses. TLR7 is present on the surface of cells, including type I HIV hand, foot and mouth disease and influenza A viruses, including through the identification of a variety of single-stranded RNA virus, antiviral immune mediated body. TLR7 ligand-activated downstream adapter protein molecule MyD88 activates signal is passed to the downstream, the final activation of the transcription factor NF-κB and produce a variety of pro-inflammatory cytokines and type I interferons, causing the body's inflammatory response. TLR7 is a type I transmembrane protein composed of the extracellular region, transmembrane region and intracellular region. Its extracellular region by a plurality of the leucine-rich LRR area, mainly involved in TLR7 ligand recognition, transmembrane region TLR7 molecule primarily responsible for anchoring, and the intracellular region of TLR7 activation is responsible for the signal transduction process. Although the study found that the activation of the innate immune response of a variety of single-stranded RNA virus infection both TLR7 molecules are involved in, but in the HCV infection caused by the body's natural immune response, TLR7 role played unclear. In addition, although the TLR7 molecule activation signal is passed down through its intracellular region, but the TLR7 molecule intracellular region-mediated signal transduction of specific mechanisms unclear. According to the above background, the present study from HCV infection activate the body's natural immune response to start, to clarify the role of TLR7 in the HCV infection process, and further affect the function of the angle of the protein primary structure explained TLR7 is activated by intracellular region specific signaling mechanism. Therefore, this study TLR7 molecule's role and the TLR7 molecular structure of the intracellular region signaling two aspects from HCV infection process is divided into the two parts as follows: 1 TLR7 molecule HCV infection process in the study of the role of first using stable transfection of the HCV genome of the Huh7 cells by the expression of the HCV genome group RNA stimulate peripheral blood mononuclear cells (PBMCs) from, by ELISA detecting cytokine expression amount, we found that HCV genomic RNA able to stimulate PBMCs significantly with the upregulation of TNF-alpha and of IFN- α. Thus confirmed that the body's natural immune response to HCV can be activated through their single-stranded RNA genome. Since the natural state, TLR7 ligand is a single stranded RNA virus, so that the activation is likely mediated through TLR7. Since the viral genome in the inner body of the cell will be degraded into oligonucleotide fragments of many different sizes (ORNs) according TLR7 recognition sequence characteristics HCV genome as a template synthetic multiple ORNs and stimulation with these ORNs new separation of PBMCs cells by ELISA, we confirmed the expression of TNF-α and IFN-α the 1000 ORN1000 raised located HCV 3 'terminal the tail ORNHCVtail able to stimulate PBMCs. TLR7 ligand-activated after the activation of the transcription factor NF-κB and IRF7, which trigger the expression of genes. Therefore, we use the synthetic ORNs stimulate Huh7 cells by dual luciferase reporter gene assay, we found ORN1000, and ORNHCVtail can activate NF-κB and IRF7. In order to determine the TLR7 whether it is involved in the immune activation caused by the HCV infection process by RNA interference, we found that when TLR7 interference ORNs stimulated cells caused by the activation of NF-κB intensity decreased significantly. Through TLR7 overexpression plasmid transfection, the NF-κB activation strength improved significantly. Therefore, we determined the HCV genomic fragment for the activation of the body's natural immune response through TLR7. 2. TLR7 molecular structure of the signal transfer TLR7 molecules are activated through a series of signal transduction pathways to activate gene expression. TLR7 by intracellular region recruit the adapter protein MyD88 to activate the signaling pathway, the molecule, however, specifically through the the intracellular region which sites mediated MyD88 raising inconclusive. Thus, the intracellular TLR7 zone protein primary structure analysis, in order to filter out the TLR7 signal transduction plays a key role loci, and explain the role of which generates mechanisms. First, we analyzed the sequence features of intracellular TLR7, and constructed the eukaryotic expression plasmid of full-length and half-length of the intracellular region of the mutant, through the activation of NF-κB dual luciferase reporter gene assay, we found transfected with the experimental group of the the TLR7 intracellular region of full-length and half-length deletion mutants transfected with empty plasmid negative control group were no difference between key sites, so we will TLR7 intracellular region locked in the intracellular region after half segment. Next, in the second half after the intracellular region of about 20 amino acids for the interval constructed a series of deletion plasmids. By detecting the activation of NF-κB, we lock key sites in a region containing 16 amino acids. Then, through a series of site-specific point mutations, that these 16 amino acids in the presence of 1004 and 1006 are located in the two arginine can impact TLR7 function. Finally, we proved by co-immunoprecipitation method, 1004 two arginine arginine affect TLR7 signaling through the influence of a combination of TLR7 downstream adapter protein MyD88. In summary, this study first confirmed HCV infection caused by the natural immune response mediated by TLR7 molecule lack of TLR7 molecule activation signal will cause the body to significant decrease in the level of immune response against HCV infection. Further analysis showed that the molecular structure and function of TLR7 area is located in the intracellular that TLR7 arginine 1004 through a combination of impact the TLR7 and downstream adapter protein MyD88 way affect TLR7 activation of the signal transduction process. This finding further elucidate the molecular basis of the TLR7 signaling mechanisms, and provide a new target is based on a TLR7 clinical diagnosis and treatment.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis C (non- A non-B )
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