The changes in B-cell growth induced by EBV infection are thought to mimic those that occur during the natural process of B-cell activation and differentiation (67
). Two of the key natural signals for B-cell activation, namely, ligand interactions with the CD40 receptor and the BCR, are supplied by the constitutive signaling activity of the EBV LMP1 and LMP2 proteins. We now provide evidence that a third proliferative signal provided by TLR signaling (56
) is induced by EBV infection and contributes to the initial B-cell growth response.
Contact between naive B cells and UV-inactivated EBV upregulated the expression of a number of interferon-responsive genes, including those for TLR7 and MyD88. The virus-cell interaction provoking the initial interferon response was not pursued here. However, the described stimulation of TLR2 by HSV, CMV, and varicella-zoster virus glycoproteins suggests that stimulation of TLR2, which is expressed on the plasma membrane of B cells, is a good candidate. TLR7 was the only TLR observed to be upregulated and, in contrast TLR9 expression, was downregulated by contact with untreated or UV-inactivated EBV. The repression of TLR9 initially seems surprising since the HSV and CMV herpesviruses stimulate TLR9 signaling through their CpG-rich genomes (39
) and stable expression of LMP2A in B cells of transgenic mice moderately upregulated TLR9 expression in a BCR-dependent manner (70
). NF-κB p65 suppresses TLR9 transcription (66
). EBV binding to the CD21 cell surface receptor activates NF-κB (62
), and a principal function of LMP1 is NF-κB activation. Thus, EBV-induced NF-κB activity may be the source of the TLR9 downregulation. TLR7 and TLR9 also modulate each other's activity. TLR7 signaling itself mediates NF-κB activation, creating a negative feedback loop for TLR9 expression, and conversely, TLR9 can physically interact with TLR7 and inhibit TLR7 function in a dose-dependent manner (71
). MyD88 is an adaptor molecule that, upon TLR stimulation, is recruited to the cytoplasmic tail of all TLRs except TLR3 (17
) and is necessary for the subsequent recruitment of the tumor necrosis receptor-associated factors TRAF3 and TRAF6 and the kinases IRAK4 and IRAK1. The upregulation of TLR7 and its adaptor MyD88, in concert with the downregulation of TLR9, would establish conditions suited for TLR7 pathway activity.
Naive B cells, the target of EBV infection in vivo, have low TLR expression compared to memory B cells. Proliferation of naive B cells in response to TLR7 ligand required costimulation with alpha interferon to upregulate TLR7 expression (7
). We also saw no stimulation when naive B cells were treated with the TLR7 ligand R837 alone, but naive B-cell growth was stimulated by the combination of R837 and UV-inactivated EBV, presumably because of the induction of TLR7 and MyD88 as part of the interferon-stimulated gene response that followed initial contact between EBV and the B cell. A contribution of TLR7 activity to initial B-cell outgrowth was supported by the reduced growth observed when the EBV-infected naive B-cell cultures were treated with IRS 661, a TLR7-specific inhibitory oligonucleotide. While TLR7 expression is upregulated by EBV contact with the cell in the absence of viral gene expression, EBV gene expression may be a source of the single-stranded RNA that normally activates TLR7 signaling.
Two interferon regulatory factors, IRF-5 and IRF-7, are recruited to MyD88 and activated by TLR7 signaling. IRF-7 was first described in type III latently infected B cells, where it was identified as a transcription factor that bound and repressed the EBV Qp promoter that serves as an alternative promoter for driving EBNA1 expression (75
). Subsequently, LMP1 induction and activation of IRF-7 and a positive feedback loop of IRF-7 induction of LMP1 were described previously (52
). IRF-7 activation leads to nuclear translocation and induction of all of the alpha interferon gene promoters, as well as the beta interferon gene promoter (25
). Despite this activity, EBV has been able to incorporate IRF-7 into its own biology in type III latently infected cells. We found that IRF-5 expression was also induced by EBV infection. Stimulation of TLR7 signaling induces IRF-5 (58
), and we noted that IRF-5 induction after infection of 4E3 B cells coincided temporally with the time at which TLR7 was upregulated. IRF-5 was initially identified as a transcription factor expressed as multiple splice variants that is active on type I interferon promoters (5
mice have a reduced type I interferon response to HSV type 1 infection and enhanced viral propagation (72
). Alpha and beta interferon production in response to TLR7 stimulation is also impaired in Irf5−/−
). IRF-5 has also been recognized as a key mediator of cytokine induction after TLR stimulation and has been proposed to activate cytokine promoters synergistically with NF-κB (49
). While an initial induction of cytokines such as interleukin-6 and -12 may be beneficial for EBV infection, continued interferon production and IRF-5 activity would be expected to have a negative impact on EBV-induced B-cell expansion. For example, IRF-5 induces apoptosis in response to DNA damage (72
) and BJAB B cells stably expressing IRF-5 formed fewer and smaller tumors in mice than did control BJAB cells (4
). The continued expression of IRF-5 in type III cell lines therefore suggested that, as is the case for IRF-7, EBV must modulate IRF-5 activity to balance the positive and negative downstream consequences. Initially to test this hypothesis, EBV infection of naive B cells was performed in the presence of a 10-fold dose of R837 to overstimulate the TLR7 pathway. R837 at 20 μg/ml completely blocked B-cell growth when supplied concurrently with virus infection. However, if EBV latent gene expression was allowed to initiate prior to the addition of the R837, then there was no negative effect on growth compared to that of untreated, EBV-infected B cells, implying that one or more of the EBV latency gene products was modifying signaling downstream of TLR7.
We explored ways in which IRF-5 activity could be modified in EBV-infected cells and obtained evidence for two separate mechanisms. IRF-5 is expressed as multiple splice variants, and alterations in IRF-5 splicing have been linked to the development of systemic lupus erythematosus, suggesting that different splice variants may differ in their functional properties (22
). An examination of IRF-5 splice variant expression in EBV-infected cells led to the identification of a novel IRF-5 splice variant, V12, that was induced by EBV infection and was expressed in all type III latently infected cells expressing IRF-5. The V12 sequence encoded a truncated IRF-5 protein that retained the DNA binding domain but lacked the nuclear export signal and transactivation domain. In transient expression assays, V12 was constitutively nuclear and negatively regulated IRF-5 transactivation of an alpha interferon promoter. The amount of V12 IRF-5 mRNA in LCL cells was 6.9-fold less than the sum of the other IRF-5 transcripts, as measured by quantitative RT-PCR (data not shown). However, the V12 protein is constitutively nuclear and by occupying IRF binding sites on responsive promoters could attenuate binding by IRF-5 entering the nucleus in response to signaling. A non-DNA binding, activation domain-only IRF-5 protein that acted as a functional negative mutant form in reporter assays has previously been described (47
), as have splice variants of IRF-1 (41
), IRF-2 (37
), and IRF-3 (31
) that have altered functional properties.
We further observed that IRF-4 was consistently expressed in type III cell lines expressing IRF-5. IRF-4 negatively modulates IRF-5 activity after TLR stimulation by competing with IRF-5 for binding to MyD88 (51
). EBV genes expressed in type III latency have been shown to upregulate IRF-4 (13
). Experiments with B-cell lines with conditional expression of EBNA2 or LMP1 revealed firstly that EBNA2 or EBNA2-regulated EBV genes were necessary for IRF-4 expression and secondly that LMP1 was one of the genes responsible for the induction. The combination of V12 and IRF-4 induction would provide negative regulation at both the IRF-5 protein activation and IRF-5 promoter upregulation steps. Further, since IRF-4 does not compete with IRF-7 binding to MyD88, the induction of IRF-4 could bias the downstream signal from TLR7 activation away from an IRF-5 gene expression signature and toward an IRF-7 signature.
The extent to which IRF-5 is integrated into the regulation of EBV gene expression remains to be investigated. The only reports to date have shown that IRF-5 heterodimerization with IRF-7 negatively affects IRF-7 activation of LMP1 expression (53
) and that IRF-5 is able to downregulate a promoter for the BamHI-A rightward transcripts (14
) that generate EBV-encoded microRNAs (54
). The induction early after infection of TLR7 and the TLR7 downstream adaptor MyD88 and effector IRF-5, along with the effects of the TLR7 ligand R837 and the TLR7 inhibitor IRS 661 on the proliferation of EBV-infected naive B cells, indicates that EBV uses TLR7 signaling to promote the initial phase of B-cell activation and expansion. The induction of the negative regulators V12 IRF-5 and IRF-4 implies that subsequent modulation of the TLR7 pathway is necessary for successful establishment of a latent infection.