Alterations in the structure of the EBV nuclear antigen, EBNA1, in epithelial cell tumours

The EBV nuclear antigen, EBNA1, is the only viral protein consistently expressed in all virus-infected cells. It is required in trans for viral replication, maintenance of EBV extrachromosomal episomes, and transcriptional transactivation in latently-infected B-cells. It binds RNA suggestive of a regulatory role in post-transcriptional events and in transgenic mice, it is tumorigenic. In RNase protection studies relating to the EBV-associated tumour, nasopharyngeal carcinoma (NPC), we show that a C-terminal EBNA1 RNA probe from the prototype B95-8 marmoset strain can protect its own mRNA from enzymatic digestion, but does not fully protect EBNA1 mRNA from NPC cells. This finding is consistent with changes in the coding region for the antigen. We thus determined the sequences of EBNA1 genes derived from an NPC xenograft and numerous patient biopsies and identified a number of mutations in the gene in these human cells, relative to B95-8. Many of the nucleotide changes would lead to non-conservative amino acid alterations in apparently functionally significant regions of the protein. We show that although some of the mutations lie in regions designated as critical to DNA binding, they have negligible effect on this property of EBNA1. The basic regions in EBNA1 that may bind to RNA, at least in vitro, are exempt from mutation. Thus, unless the alterations are 'silent', which for such a critical viral function seems unlikely, they may relate to as yet unmapped viral activities, such as a role in tumorigenesis and the ability of EBNA1 to evade the cellular immune system, or be associated with the ability of the antigen to regulate gene transcription

Widespread sequence variation in Epstein-Barr virus nuclear antigen 1 influences the antiviral T cell response

Epstein‐Barr virus (EBV) nuclear antigen (EBNA) 1 is perhaps the most widely studied EBV protein, because of its critical role in maintaining the EBV episome and its expression in all EBV‐associated malignancies. Much of this research has focused exclusively on the EBV wild‐type (wt) strain (B95‐8). Sequence analysis of the gene encoding for EBNA1 in EBV isolates from 43 Caucasians has now revealed considerable EBNA1 sequence divergence from the EBV wt strain in the majority of isolates from this population group. Importantly, T cell recognition of an endogenously processed HLA‐B8–binding EBNA1 epitope was greatly influenced by this sequence polymorphism

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein–Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP