Epstein-Barr virus BRLF1 inhibits transcription of IRF3 and IRF7 and suppresses induction of interferon-beta

Abstract

Activation of interferon regulatory factors (IRFs) 3 and 7 is essential for the induction of Type I interferons (IFN) and innate antiviral responses, and herpesviruses have evolved mechanisms to evade such responses. We previously reported that Epstein-Barr virus BZLF1, an immediate-early (IE) protein, inhibits the function of IRF7, but the role of BRLF1, the other IE transactivator, in IRF regulation has not been examined. We now show that BRLF1 expression decreased induction of IFN-beta, and reduced expression of IRF3 and IRF7; effects were dependent on N- and C-terminal regions of BRLF1 and its nuclear localization signal. Endogenous IRF3 and IRF7 RNA and protein levels were also decreased during cytolytic EBV infection. Finally, production of IFN-beta was decreased during lytic EBV infection and was associated with increased susceptibility to superinfection with Sendai virus. These data suggest a new role for BRLF1 with the ability to evade host innate immune responses.

Figure 1. EBV Rta regulates IFN-β promoter activity

A) 293T cells were transfected with EBV Rta or vector control along with IFN-β reporter plasmid and Renilla luciferase plasmid. Cells were mock-infected or infected with Sendai virus (50 HA units/ml) 16 hours post transfection. Eight hours later (24 hours post transfection), cells were harvested for firefly luciferase assays and normalized to Renilla luciferase expression. B) 293T cells were transfected with EBV Rta or vector control and Sendai virus infected 16 hours post transfection. 24 hours post transfection, cells were collected and RNA harvested for semi-quantitative RT-PCR to examine IFN-β and GAPDH expression. 293T cells were transfected with the vector control or C) IRF3 or D) IRF7 in the presence or absence of EBV Rta.or the vector control and luciferase assays were performed 24 hours post transfection. Data are shown as the fold change (relative to vector control) ± standard deviation of experiments performed in triplicate.

Figure 2. EBV Rta negatively regulates IRF3 and IRF7 protein expression but does not alter IRF5 expression

293T cells were transfected with Flag-IRF3, Flag-IRF5, Flag-IRF7, and/or Flag-EBV Rta as indicated. A) 48 hours post transfection whole cell lysates were collected and Western blot analyses performed examining Flag expression/exogenous protein expression. B) 48 hours post transfection, cytoplasmic extracts and nuclear extracts were collected and Western blot analyses performed examining Flag expression in the two extracts. Tubulin and lamin were used as loading controls.

Figure 3. All classified EBV Rta function domains are required for the regulation of IRF3 and IRF7 expression

293T cells were transfected with Flag-IRF3, Flag-IRF7, and GFP-EBV Rta or select GFP-EBV Rta mutants as indicated. 48 hours post transfection whole cell lysates were collected and Western blot analyses performed examining Flag and GFP expression. Tubulin was used as a loading control.

Figure 4. EBV Rta reduces endogenous RNA levels of IRF3 and IRF7

293T cells were transfected with vector control or EBV Rta as indicated. 14 hours post transfection, cells were A) stimulated with 500U/ml of Type I IFN or B) infected with Sendai virus (50 HA units/ml). Cells were collected and RNA harvested 24 hours post tranfection. Semi-quantitative RT-PCR was performed to detect expression of IRF3, IRF5, and IRF7. Actin and GAPDH were used as loading controls.

Figure 5. Endogenous IRF3 and IRF7 RNA and protein expression is reduced during EBV lytic infection

Viral reactivation was performed in Akata cells where the cells were incubated with F(AB′)2 fragment to human IgG for 0 hours (0 h), 24 hours (24 h), or 48 hours (48 h). A) Total RNA was harvested and semi-quantitative RT-PCR performed examining IRF3, IRF5, IRF7, and Rta RNA expression. B) Whole cell lysates were collected and Western blot analyses performed examining IRF3, IRF5, IRF7, Rta, and EA-D protein expression. Densitometry was performed in which relative RNA or protein expression was normalized to relative GAPDH expression. Data are shown as fold change (relative to 0 hour post induction) ± standard deviation of experiments performed in triplicate.

Figure 6. Endogenous IFN-β RNA levels decrease during EBV lytic infection and coincide with increased susceptibility to Sendai virus infection

Viral reactivation was induced in Akata cells as in Figure 5. A) Total RNA was harvested and semi-quantitative RT-PCR performed to detect IFN-β and GAPDH RNA at 0, 24, and 48 hours post induction. Densitometry was performed where relative IFN-β levels were normalized to relative GAPDH levels. Data are shown as the fold change (relative to 0 hour post induction) ± standard deviation of experiments performed in triplicate. B) 24 hours after induction (or mock-induction), Akata cells were infected with Sendai virus (50 HA units/ml). 24 hours after infection, supernatant fluids were collected for plaque assays on LLC-MK₂ cells. Viral titers were determined and data are shown as PFU/ml ± standard deviation of experiments performed in triplicate.