Cellular Immediate-Early Gene Expression Occurs Kinetically Upstream of Epstein-Barr Virus bzlf1 and brlf1 following Cross-Linking of the B Cell Antigen Receptor in the Akata Burkitt Lymphoma Cell Line

Abstract

The Epstein-Barr virus (EBV) lytic activator genes bzlf1 and brlf1 are conventionally referred to as immediate-early (IE) genes. However, previous studies showed that the earliest expression of these genes was blocked by cycloheximide when the EBV lytic cycle was induced by histone deacetylase (HDAC) inhibitors and protein kinase C agonists. Anti-IgG activates a complex signal transduction pathway that leads to EBV lytic activation in the Akata cell line. Here we demonstrate that in Akata cells, where lytic cycle activation occurs very rapidly after anti-IgG treatment, de novo protein synthesis is also required for induction of bzlf1 and brlf1 expression. New protein synthesis is required up to 1.25 h after application of anti-IgG; bzlf1 and brlf1 mRNAs can be detected 1.5 h after anti-IgG. Five cellular IE genes were shown to be expressed by 1 h after addition of anti-IgG, and their expression preceded that of bzlf1 and brlf1. These include early growth response genes (egr1, egr2, and egr3) and nuclear orphan receptors (nr4a1 and nr4a3). These genes were activated by anti-IgG treatment of Akata cells with and without the EBV genome; therefore, their expression was not dependent on expression of any EBV gene product. EGR1, EGR2, and EGR3 proteins were kinetically upstream of ZEBRA and Rta proteins. Expression of EGR1, ZEBRA, and Rta proteins were inhibited by bisindolylmaleimide X, a selective inhibitor of PKC. The findings suggest a revised model in which the signal transduction cascade activated by cross-linking of the B cell receptor induces expression of cellular IE genes, such as early growth response and nuclear orphan receptor genes, whose products, in turn, regulate bzlf1 and brlf1 expression.Two Epstein-Barr virus (EBV) genes, bzlf1 and brlf1, encode protein products, ZEBRA and Rta, that control the transition from viral latency to lytic replication (12,13,23,34,46). These two proteins are activators of viral and cellular gene expression, play essential roles in lytic viral DNA replication, and impinge on many host cell processes, such as signal transduction, cell cycle control, and cellular senescence (10,27,30,31). The bzlf1 and brlf1 genes are not expressed during latency, but all external stimuli known to activate the EBV lytic cascade induce their expression (3,16,28,39,48). Therefore, a central question is, "What controls the expression of these two EBV lytic activator genes?" We refer to this question as the "upstream problem" in lytic activation (29,44).A full understanding of the upstream events has been elusive for several reasons. Many mechanically heterogeneous stimuli activate the lytic cascade in cultured lymphoid cells, where the molecular events can be readily analyzed. Each cell line seems to respond to the inducing stimuli in an idiosyncratic fashion (3,16,28,39,48). The lytic cycle-inducing agents vary in the duration of exposure required to elicit a response. Moreover, the cells vary in their response time (11). It is not yet known whether the many pathways engaged by the diverse sti...

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Role of RNF4 in the Ubiquitination of Rta of Epstein-Barr Virus

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