Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells

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 ( 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 IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication.

Deep Proteomic Deconvolution of Interferons and HBV Transfection Effects on a Hepatoblastoma Cell Line

“…In order to dissect the IFN-response in HepG2 cells and to compare the IFN-response to an HBV-transfection induced state, we performed two experiments, with five replicates in each: HepG2 cells with/without IFN-I or IFN-III stimulation (IFN-α2 or IFN-λ3) and a comparison of HepG2 cells versus HepG2.2.15 cells; in addition, we also included our previously published work: 12 HepG2.2.15 cells with/without IFN-I or IFN-III stimulation, into our bioinformatic analysis ( Figure 1 ).…”

“…Thus, in the IFN-I/III conditions, 12 and 7% of all proteins showed altered expression. Note that the MTT assays in our previous work 12 were used to establish IFN treatment concentrations that were not detrimental to cell survival, so the above-mentioned differential results cannot be interpreted as alterations that would be expected because of cell death. Not surprisingly, proteins identified as upregulated/downregulated under IFN-I treatment strongly overlapped with those upregulated/downregulated under IFN-III treatment ( P = 10 –107 / P = 10 –46 ).…”

“… 79 However, these proteins were sharply downregulated in HepG2.2.15 cells, with P < 10 –7 in both cases. Such discrepancies may indicate the HBV stage of infection that is predominant in HepG2.2.15 cells, with HBV proteins found near the limits of MS detection, 12 as well as the extreme levels of the HBx protein present in an overexpression context. In fact, contradictory pro- and antiapoptotic functions have been noted with respect to HBx, 80 with speculation that such behavior relates to HBx abundance in infected cells.…”

“… 9 Another deep proteomic analysis of IFN-γ effects on mesenchymal stromal/stem cells 10 has been conducted, but such results would not be expected to be good proxies for IFN-I effects, given the differences in pathways that are triggered upon type I or II stimulation. 11 We previously conducted a high-depth analysis of IFN type I and III effects on a human hepatoblastoma cell line transfected with HBV (HepG2.2.15), with the aim of testing potential therapeutic effects; 12 however, this setting would not be ideal for the study of authentic IFN effects, as viral versus IFN stimulation of cells might well overlap upon analysis of altered protein expression.…”

Deficiency of STING Promotes Collagen-Specific Antibody Production and B Cell Survival in Collagen-Induced Arthritis

“…Dimethyl labeling and LC-MS/MS analysis were performed as described in Makjaroen et al (28). In brief, B cells were isolated from Stinggt/gt and WT mice after injection with CFA and CII for 3 weeks.…”