Interferon Regulatory Factor 4 (IRF-4) Targets IRF-5 to Regulate Epstein-Barr Virus Transformation

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdownmediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation

Interferon Regulatory Factor 4 (IRF-4) Targets IRF-5 to Regulate Epstein-Barr Virus Transformation

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdownmediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation

Toll-Like Receptor 7 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus. Toll-like receptor 7 (TLR7) is involved in host innate immunity against pathogens, and its aberrant activation is linked to the development of systemic lupus erythematosus (SLE, also called ‘‘lupus’’). Type I interferons (IFN) are apparently driving forces for lupus pathogenesis. Previously, we found that EBV latent membrane protein 1 (LMP1) primes cells for IFN production. In this report, the relationship among EBV LMP1, TLRs, and IFN production are examined. We find that TLR7 activation increases the expression of EBV LMP1, and IFN regulatory factor 7 (IRF7) is involved in the stimulation process. TLR7 activation did not induce IFNs from EBV-infected cells, but potentiates those cells for IFN production by TLR3 or TLR9 activation. In addition, we find that LMP1 and IFNs are coexpressed in the same cells in some lupus patients. Therefore, the aberrant activation of TLR7 might induce LMP1 expression and LMP1-expression cells may be producing IFNs in lupus patients. These results suggest EBV might be an exacerbating factor in some lupus patients via promoting IFN production

EBV lytic replication plays limited role in the induction of LMP1.

<p>A. TLR7 activation induces LMP1 in EBV-transformed cells defective for viral lytic replication. An EBV-BRLF-null virus (RKO) transformed primary B cell line (LCL-RKO) and its parental virus transformed cells (LCL-wtEBV) were treated with imiquimod overnight. Cell lysates were used for Western blot analysis with LMP1 and tubulin antibodies. The identity of proteins is as shown. B. TLR7 activation failed to induce EBV lytic replication in EBV-transformed cells. LCL-wtEBV and LCL-RKO cells were treated with imiquimod overnight. The positive control was Akata cells treated with anti-human IgG. Cell lysates were used for Western blot analysis with LMP1 and Tubulin antibodies. The identity of proteins is as shown.</p

Toll-Like Receptor 7 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1

<div><p>Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus. Toll-like receptor 7 (TLR7) is involved in host innate immunity against pathogens, and its aberrant activation is linked to the development of systemic lupus erythematosus (SLE, also called “lupus”). Type I interferons (IFN) are apparently driving forces for lupus pathogenesis. Previously, we found that EBV latent membrane protein 1 (LMP1) primes cells for IFN production. In this report, the relationship among EBV LMP1, TLRs, and IFN production are examined. We find that TLR7 activation increases the expression of EBV LMP1, and IFN regulatory factor 7 (IRF7) is involved in the stimulation process. TLR7 activation did not induce IFNs from EBV-infected cells, but potentiates those cells for IFN production by TLR3 or TLR9 activation. In addition, we find that LMP1 and IFNs are co-expressed in the same cells in some lupus patients. Therefore, the aberrant activation of TLR7 might induce LMP1 expression and LMP1-expression cells may be producing IFNs in lupus patients. These results suggest EBV might be an exacerbating factor in some lupus patients via promoting IFN production.</p> </div

TLR7 activation stimulates the expression of EBV LMP1.

<p>A. TLR7 agonist stimulates EBV LMP1. SavIII or IB4 cells were treated with TLR7 agonist (imiquimod; 25 µg/ml), TLR3 agonist (poly (I:C); 10 µg/ml) or TLR9 agonist (ODN2395; 2.5 or 5 µM) for 24 hours. Cell lysates were used for Western blot analysis with LMP1 and GAPDH antibodies. The identity of proteins is as shown. B. TLR7 activation increases detectable LMP1-postive cells. IB4 cells were treated with imiquimod (25 µg/ml), for 24 hours, and the cells were fixed and stained with LMP1 primary and Alexa Fluor 647-labeled secondary antibodies. DAPI was used to stain the nuclei. The images were captured under identical conditions. The colors were artificially mounted to facilitate viewing. Blue, nuclei; red, LMP1. C. Quantification of detectable LMP1-positive cells. IB4 cells were treated and stained as in Panel B. The percentages of LMP1-positive cells were counted in 10 randomly selected fields. For untreated controls, the average number of cells per field with standard deviation is 201.5±59.5; while in imiquimod treated cells, the average number of cells per field with standard deviation is 170.6±70.8. The difference is statistically significant (<i>p</i><0.01). The <i>p</i> value was calculated by paired Student's t test with the use of Microsoft Excel.</p

EBV LMP1 is localized in the IFN-producing cells in lupus patients.

<p>PBMCs from healthy or lupus patients were stained with IFN and LMP1 antibodies. Alexa Fluor 488- and Alexa Fluor 647-labeled secondary antibodies were used to distinguish the signals from IFN and LMP1, respectively. DAPI was used to stain the nuclei. The colors were artificially mounted to facilitate viewing: blue, nuclei; green, IFN; red, LMP1. Panel A: A representative result for apparent healthy individual is shown. Panel B: Result for lupus patients (Patient #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043317#pone-0043317-t001" target="_blank">Table 1</a>) is shown. Circle: dual-positive cells. Arrow: LMP1 single positive cell. Square: IFN- single positive cell. Panel C: Healthy individual #5. Arrow indicates an IFN-positive cell. Some red blood cells are presents (no nuclei); Panel D: Result for lupus patients #11: arrows indicate the IFN-positive cells; Panel E: DG75 cells (negative control); Panel F: IB4 cells infected with Sendai virus (positive control). Panels: C–F: a, IFN only; b, LMP1 only; c, IFN plus LMP1; and d, IFN, LMP1, and nuclei.</p

Potential exacerbating role of EBV in lupus pathogenesis via IFNs.

<p>EBV infection increases the expression of TLR7. EBV-infected cells may respond to TLR7 stimulators present in lupus patients, and increase the expression levels of LMP1; LMP1 primes the cells for IFN production; TLR3 and TLR9 stimulators induce IFN production in those EBV-infected cells. High amounts of IFNs would be responsible for more auto-antibody productions, cell/tissue damages, and eventually more self-nucleotide complexes containing various TLR activators, including for TLR7; TLR7 activation again may promote more LMP1 expression.</p