Viral induction of AID is independent of the interferon and the Toll-like receptor signaling pathways but requires NF-kappaB

Abstract

Activation-induced cytidine deaminase (AID) is expressed in germinal centers of lymphoid organs during immunoglobulin diversification, in bone marrow B cells after infection with Abelson murine leukemia retrovirus (Ab-MLV), and in human B cells after infection by hepatitis C virus. To understand how viruses signal AID induction in the host we asked whether the AID response was abrogated in cells deficient in the interferon pathway or in signaling via the Toll-like receptors. Here we show that AID is not an interferon responsive gene and abrogation of Toll-like receptor signaling does not diminish the AID response. However, we found that NF-kappaB was required for expression of virally induced AID. Since NF-kappaB binds and activates the AID promoter, these results mechanistically link viral infection with AID transcription. Thus, induction of AID by viruses could be the result of several signaling pathways that culminate in NF-kappaB activation, underscoring the versatility of this host defense program.

Figure 1.

AID up-regulation does not require intact IFN signaling. (A) Mo-MLV infection of mouse bone marrow results in AID upregulation. CD79b expression is a loading control for the RT-PCR reaction. (B) IFN-γ and IFN-αR–deficient bone marrow cells induce AID expression upon viral infection.

Figure 2.

AID is up-regulated in bone marrow B cells after ligation of endosomal TLRs. (A) AID expression after transfection of BM cells with CpG for 24 h. (B) Time-course of AID expression after transfection of BM cells with double strand RNA.

Figure 3.

Abrogation of TLR signaling does not diminish the expression of virally induced AID. (A) Bone marrow cells from MyD88^−/− and MyD88^−/−Trif^−/− mice induce AID expression upon infection with Ab-MLV. (B) B cells deficient in TLR signaling are susceptible to transformation by Ab-MLV. (C) Mice deficient in MyD88 are as susceptible as their wild-type counterparts to death after Ab-MLV infection.

Figure 4.

NF-κB is required for expression of virally induced AID. (A) Bone marrow cells from NF-κB (p50)^−/− mice cannot induce AID expression upon infection with Ab-MLV. (B) NF-κB (p50)^−/− splenic B cells are defective in CSR. Levels of CSR to IgG1 and IgG3 are shown 72 h after stimulation with LPS and IL-4 or CD40 and IL-4. (C) NF-κB is required for optimal AID expression in switching B cells.

Figure 5.

NF-κB–p50 is recruited to the mouse AID promoter to directly activate AID expression during infection by Ab-MLV. (A) Occupancy of the NF-κB binding site present in the AID promoter is assessed by ChIP before (day 0) or during (day 6) infection of wild-type or NF-κB–p50–deficient animals. Occupancy of an NF-κB binding site present in the nearby APOBEC1 promoter was evaluated as a negative control. Threefold titrations are shown for input, anti–NF-κB immunoprecipitation, and isotype control immunoprecipitation samples. (B) AID promoter activity in infected cells, in the presence or absence of the κB binding site. Bone marrow cells were coinfected with Ab-MLV and with a retroviral luciferase reporter construct containing either the wild-type κB site (wt κB Luc) or a mutated κB site (mut κB Luc). Promoter activity for each construct was measured in relative light units per μg of protein lysate. Results are means ± SD (n = 3).