Structure and Coding Content of CST (BART) Family RNAs of Epstein-Barr Virus

Abstract

CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers.Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open reading frames (ORFs) suggest that, in addition to the much studied BARF0 ORF, there may be important products made from some of the upstream ORFs in the CST RNAs. Potential biological functions are identified for two of these. The product of the RPMS1 ORF is shown to be a nuclear protein that can bind to the CBF1 component of Notch signal transduction. RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2. The protein product of another CST ORF, A73, is shown to be a cytoplasmic protein which can interact with the cell RACK1 protein. Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, the results suggest a possible role for CST products in growth control, perhaps consistent with the abundant transcription of CST RNAs in cancers such as nasopharyngeal carcinoma.In several types of infection, in addition to the well-established EBNA, LMP and EBER genes, Epstein-Barr virus (EBV) has been found to express various spliced RNAs transcribed rightward from the region spanning 150,000 to 161,000 on the B95-8 EBV genetic map. These have been referred to as complementary strand transcripts (CSTs), BamHI A rightward transcripts (BARTs), or the BARF0 RNAs. RNAs of this type were originally identified in cDNA made from the C15 xenograft culture of nasopharyngeal carcinoma (NPC) tissue (17). Similar RNAs were subsequently found in various EBV-positive NPC tumor biopsies and xenografts, Burkitt's lymphoma, lymphoid cell lines (LCLs) (3,6,11,15,19,31,43), and biopsies of oral hairy leukoplakia (24). Expression of CST RNAs has also been demonstrated in peripheral blood of normal human carriers of EBV (5), sera from NPC patients have been found to immunoprecipitate a protein product of the BARF0 open reading frame (ORF) made in vitro (12), and cytotoxic T lymphocytes that respond to a peptide derived from BARF0 have been identified in EBV-infected people (22).A very complicated picture of alternatively spliced CST RNAs has built up (36, 38), but some of the proposed structures have been deduced using very sensitive reverse transcription (RT)-PCR methods or have been only single isolates from cDNA libraries, and thus they may yet represent very minor species within the family of RNAs that can be expressed. Predominant sizes of the RNAs expressed have been deduced from Northern blots, but these have only partly been related to the spliced RNA structures. In addition, the coding content of the CST RNAs remains uncertain. Most attention has focused on the BARF0 ORF, particularly a sp...

Diversity of Epstein–Barr virus BamHI-A rightward transcripts and their expression patterns in lytic and latent infections

“…The BARTs start site is thought to be just upstream of Exon I, and all the transcripts utilize the same polyadenylation signal, almost at the end of Exon VII (Smith et al, 2000). The B95-8 strain has also been reported to generate BARTs, despite the lack of Exons II, III and IV, and our data were consistent with that result, except for the RB3 form (Sadler …”

“…3). These data suggest the possibility that there are unidentified splicing forms of BARTs and the related proteins in addition to the six known BARTs, as pointed out elsewhere (Chen et al, 1999;Smith et al, 1993Smith et al, , 2000Sadler & RaabTraub, 1995;Smith, 2001). …”

“…Six types of BART splicing forms have been reported to date (Chen et al, 1999;Smith et al, 1993Smith et al, , 2000Sadler & Raab-Traub, 1995;Smith, 2001) (Fig. 1, Table 2).…”

“…Functional analyses have demonstrated that the BARTs might serve as regulatory RNAs to limit lytic gene expression, and help ensure maintenance of the viral latency program (Karran et al, 1992). The six BARTs are also assumed to possess their own ORFs, and their functions have been reported (Zhang et al, 2001;Smith et al, 2000;Fries et al, 1997). Thus, the presence of BART proteins has been assumed in EBV-infected cells, although not all the proteins have been identified in vivo.…”

Hypothesis: a novel route for immortalization of epithelial cells by Epstein-Barr virus

“…If one hypothesizes that an EBV protein might bind DNA near or at telomere sites and prevent their shortening, the question would be which of the viral proteins, within p31 DNA, might ful®l this role? Candidate genes may be found among those associated with the epithelial cell tumours, such as NPC (Hitt et al, 1989;Chen et al, 1992;Brink et al, 1998;Zhang et al, 1998;Hayes et al, 1999;Xue et al, 2000;Smith et al, 2000), see Figure 1. There is no suggestion in the literature (Strockbine et al, 1998) or in its structure (Baer et al, 1984), that BARF1, the only p31-containing viral function known to stimulate cell growth (Wei and Ooka, 1989;Wei et al, 1994Wei et al, , 1997, might act in this manner.…”