Abstract
Interferon lambda (IFN-λ) is a relatively unexplored, yet promising antiviral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of antiviral proteins to multiple points of the viral life cycle, as well as facilitating the identification of antiviral proteins not previously known to be elicited upon HBV infection (e.g. IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through several essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated whereas cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation toward the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 versus IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication.
Highlights
Interferon lambda (IFN-) is a relatively unexplored, yet promising antiviral agent
To determine whether HepG2.2.15 cells respond to IFN-3, we performed qPCR to investigate the expression of the classical interferonstimulated gene (ISG), namely OAS1, MX1 and ISG15 in HepG2.2.15 cells treated with various amounts of IFN-3 for 24h
These results indicated that HepG2.2.15 cells responded to IFN-3 stimulation
Summary
Experimental Design and Statistical Rationale—Mixing of dimethyllabeled samples corresponding to IFN-3, IFN-␣2a, and control (PBS) treatments were performed at n ϭ 5 versus the typical n ϭ 3 to emphasize depth. HepG2.2.15 cells were plated at 5 ϫ 106 cells in T-75 flasks and grown in complete DMEM for 24 h at 37 °C These cells were subsequently cultured in media with 100 ng/ml of IFN-3 or 100 ng/ml of IFN-␣2a (11100 –1, pbl assay science, NJ) or PBS (control) for another 24 h. Equal amounts of protein from treated and untreated HepG2.2.15 were reduced and alkylated by dithiothreitol (DTT) treatment for 30 min at 37 °C and iodoacetamide (IA) treatment for 30 min at room temperature in the dark, respectively These samples were further quenched with DTT at least 15 min at room temperature before incubating with trypsin at a ratio of 1:50 at 37 °C overnight.
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