Differential Gene Expression Patterns of EBV Infected EBNA-3A Positive and Negative Human B Lymphocytes

Abstract

The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV–growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis.

Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

“…3A). This observation is in agreement with previous studies where LCLs derived from viruses with EBNA 3A deleted were found to express higher levels of ADAM28 than control LCLs (14). However, since only one EBNA 3A-and one EBNA 3B-expressing cell line were available for analysis, the individual effects of these proteins on ADAM28 expression requires further verification.…”

Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16 INK4A and p14 ARF expression

“…The experiments described here investigate the underlying biochemical mechanisms and significance of initial findings that conditional EBNA3A or EBNA3C inactivation in LCLs induces G 1 growth arrest, which for EBNA3C is associated with induction of CDKN2A p16 INK4A expression, consistent with the possibility that EBNA3A and EBNA3C's essential role is to repress CDKN2A p16 INK4A (5)(6)(7)(8). EBNA3A, EBNA3B, and EBNA3C likely arose as a consequence of gene triplication, after the evolution of EBNA2 and EBNALP (9).…”

“…Because conditional E3AHT inactivation in LCLs has similar effects on LCL growth and p16 INK4A is also elevated in EBNA3A knockout LCLs relative to wild-type LCLs (5,7,8), EBNA3A may also have a role in p16 INK4A and p14 ARF repression. Indeed, incubation of E3AHT LCLs in media without 4HT, resulted at day 6 in substantially reduced E3AHT, slowed cell growth, increased p16 INK4A and p14 ARF protein expression, and ∼1.4-fold significantly increased p16 INK4A and p14 ARF RNA levels ( Fig.…”

Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

“…To consider the potential overlap of EBNA3C and EBNA3A transcriptional effects, we compared EBNA3C effects on RNA levels with putative EBNA3A effects, which have been assessed by comparison of RNA levels in LCLs with RNA levels in B cells grown on feeder cells after infection with an EBNA3A-negative EBV (53). Overall, 52 genes were significantly regulated by both EBNA3C and EBNA3A, with a P value of <3 × 10 −23 ( Fig.…”