Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

Abstract

Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1,800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

Inhibition of Antigen Presentation by the Glycine/Alanine Repeat Domain Is Not Conserved in Simian Homologues of Epstein-Barr Virus Nuclear Antigen 1

“…The present study is concerned with the likely evolutionary importance of the EBNA1 GAR domain. In studies with EBV EBNA1, this domain is known not to be essential for the genome maintenance function of the protein and indeed is not required for EBV-induced B-cell transformation in vitro (15). Yet GAR-like motifs have been conserved in the EBNA1 molecules of rhesus and baboon LCVs, suggesting some other important function.…”

Episomal vectors for gene expression in mammalian cells

“…Recent data indicate that, during latent infection in vivo in proliferating cells, the total EBV chromosome does not require the replication‐initiation function of oriP. The essential role of EBNA1 would only be the maintenance of the circular EBV chromosome, almost certainly in conjunction with the extrachromosomal maintenance function of oriP [122,123]. The role of EBNA1 in replication of artificial oriP‐containing plasmids is still a matter of debate.…”

Epstein–Barr virus nuclear antigen 1‐specific CD4+ T cells directly kill Epstein–Barr virus‐carrying natural killer and T cells

“…EBNA1 also binds to OriP, ( 5 ) and is essential for EBV episome maintenance. ( 6 ) Thus, the existence of EBV DNA is tightly associated with EBNA1 expression in EBV‐infected cells.…”