Date Published: January 20, 2017
Publisher: Public Library of Science
Author(s): Takeya Tsutsumi, Kazuya Okushin, Kenichiro Enooku, Hidetaka Fujinaga, Kyoji Moriya, Hiroshi Yotsuyanagi, Hideki Aizaki, Tetsuro Suzuki, Yoshiharu Matsuura, Kazuhiko Koike, Ratna B. Ray.
The hepatitis C virus nonstructural protein NS5A is involved in resistance to the host immune response, as well as the viral lifecycle such as replication and maturation. Here, we established transgenic mice expressing NS5A protein in the liver and examined innate immune responses against lipopolysaccharide (LPS) in vivo. Intrahepatic gene expression levels of cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly suppressed after LPS injection in the transgenic mouse liver. Induction of the C-C motif chemokine ligand 2, 4, and 5 was also suppressed. Phosphorylation of the signal transducer and activator of transcription 3, which is activated by cytokines, was also reduced, and expression levels of interferon-stimulated genes, 2’-5’ oligoadenylate synthase, interferon-inducible double-stranded RNA-activated protein kinase, and myxovirus resistance 1 were similarly suppressed. Since LPS binds to toll-like receptor 4 and stimulates the downstream pathway leading to induction of these genes, we examined the extracellular signal-regulated kinase and IκB-α. The phosphorylation levels of these molecules were reduced in transgenic mouse liver, indicating that the pathway upstream of the molecules was disrupted by NS5A. Further analyses revealed that the interaction between interleukin-1 receptor-associated kinase-1 and tumor necrosis factor receptor associated factor-6 was dispersed in transgenic mice, suggesting that NS5A may interfere with this interaction via myeloid differentiation primary response gene 88, which was shown to interact with NS5A. Since the gut microbiota, a source of LPS, is known to be associated with pathological conditions in liver diseases, our results suggest the involvement of NS5A in the pathogenesis of HCV infected-liver via the suppression of innate immunity.
Hepatitis C virus (HCV) is one of the most prevalent infectious diseases worldwide. Chronic infection develops in the majority of patients, often leading to liver cirrhosis and hepatocellular carcinoma (HCC) . The HCV genome is a single-stranded positive RNA molecule encoding viral structural proteins such as core, E1, E2, and the p7 protein, and non-structural (NS) proteins such as NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The NS5A is a multifunctional phosphoprotein consisting of 447 amino acids residues. In addition to the association with the viral lifecycle, such as replication and maturation [2,3], NS5A is known to have pleiotropic effects on host cells, including regulation of host gene expression and cellular signaling pathways . NS5A is also implicated in antiviral resistance to interferon (IFN) . NS5A contains an IFN sensitivity-determining region (ISDR) and interacts with the IFN-inducible double-stranded RNA-activated protein kinase (PKR) through the ISDR, leading to the repression of PKR activity . NS5A also attenuates IFN action by inducing expression and secretion of interleukin (IL)-8, a pro-inflammatory chemokine that interferes with IFN through alterations in IFN-stimulated genes (ISG) . NS5A is also known to directly inhibit IFN-αsignaling by abrogating signal transducer and activator of transcription (STAT)-1 phosphorylation and nuclear translocation .
In this study, we generated transgenic mice expressing HCV NS5A protein in the liver and found that the mice have impaired TLR4 signaling. When the mice were injected with LPS, which binds to a cell surface receptor TLR4 and activates the TLR signaling pathway, suppressed activation of STAT3 and reduced induction of intrahepatic cytokines and ISGs were observed. The mechanism of impaired TLR signaling by NS5A is the inhibition of the direct interaction between IRAK1 and TRAF6. We previously reported that NS5A binds to MyD88, a major adaptor molecule in TLR, which inhibits the recruitment of IRAK1 to MyD88 in response to TLR ligands . Since the expression levels of NS5A in our transgenic mice are as low as those in human infected livers, the interaction of NS5A with MyD88 could not be detected (data not shown), but the observation that the interaction between IRAK1 and TRAF6 was impaired in transgenic mice suggests that NS5A may interact with MyD88 in the liver of transgenic mice.