Abstract
ABSTRACTA number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of infection with Epstein-Barr virus (EBV), which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen 2 (EBNA2) both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-Jκ. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function both in newly infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand 1 inhibits EBNA2-mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 expression from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into the lytic cycle in a B cell non-Hodgkin's lymphoma line by upregulating the cellular transcription factor Zeb2, which represses the transcription of BZLF1. These results support the concept that in vivo, cumulative signals from the microenvironment downregulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood.IMPORTANCE Experimental infection of resting B cells by Epstein-Barr virus leads to the growth transformation program of virus gene expression and the outgrowth of lymphoblastoid cell lines. Previous studies at the single-cell level revealed complex cellular and viral signaling networks regulating transcription of the viral genome. This study demonstrates that viral gene expression can also be radically altered by molecules expressed on stromal cells in the microenvironment of lymphoid tissue, specifically, Delta-like ligand 1 on stromal cells ligating Notch-2 on infected B cells. Activation of Notch interferes with the transactivation function of EBNA2, downregulates the expression of LMP1 and LMP2a, and inhibits the activation of lytic virus replication in a B cell non-Hodgkin's lymphoma line by preventing expression of BZLF1. The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression, enabling establishment of true latency in memory B cells.
Highlights
A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation
We found that BZLF1 mRNA transcription was inhibited only when the cells were cocultured on OP9-Delta-like ligand 1 (DLL1) cells; when the cells were cocultured with OP9-green fluorescent protein (GFP) cells, BZLF1 transcription was initiated at the same rate seen when the cells where removed from the stroma
Little is known about the regulation of Epstein-Barr virus (EBV) gene expression following primary infection of resting B lymphocytes in vivo, how EBV gene expression is downregulated from the latency III gene expression profile to the latency 0 profile seen in circulating resting memory B cells
Summary
A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. Activation of Notch interferes with the transactivation function of EBNA2, downregulates the expression of LMP1 and LMP2a, and inhibits the activation of lytic virus replication in a B cell nonHodgkin’s lymphoma line by preventing expression of BZLF1 The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression, enabling establishment of true latency in memory B cells. Epstein-Barr virus (EBV), which is carried by the vast majority of all adults worldwide, establishes a lifelong persistent infection through colonizing the B lymphocyte compartment as a latent infection It has been postulated [13] that EBV exploits the normal physiology of B cell differentiation to regulate its own gene expression. In addition to sharing a general feature of all herpesviruses, which is the ability to expand the virus-infected cell pool by periodic lytic replication to generate new infectious virions, EBV has the potential to expand the pool of virus-infected
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