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.

Author Summary

Epstein-Barr virus (EBV) infects primary human B cells and establishes a latent infection, which leads to permanently growing B cell cultures. These growth transformed B cells express a well defined set of latent viral genes, which are also expressed in post-transplant lymphomas of immunosuppressed patients. In a concerted action these latent viral proteins drive cellular proliferation and prevent apoptosis. For this study, recombinant Epstein-Barr virus mutants that lack the gene for the Epstein-Barr virus nuclear antigen-3A (EBNA-3A) were generated. EBNA-3A is a transcriptional modulator of gene expression. We show here that EBNA-3A deficient growth transformed B cells can be established in vitro. Our results suggest that EBNA-3A supports viability but is not absolutely essential for proliferation of the infected B cell. By virtue of the established EBNA-3A deficient cell lines, we could for the first time identify a broad array of cellular target genes controlled by EBNA-3A in EBV infected B cells. These EBNA-3A target genes will permit a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis.