Downloaded from www.microbiologyresearch.org by IP: 54.157.140.51 On: Thu, 31 Mar 2016 02:47:51 Journal of General Virology (1995), 76, 139 145. Printed in Great Britain 139 Sequence polymorphism in the Epstein-Barr virus latent membrane protein (LMP)-2 gene Pierre Busson, 1,~ Rachel Hood Edwards, 1 Thomas Tursz 2 and Nancy Raab-Traub ~* 1Department of Microbiology and Immunology and the Lineberger Cancer Research Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA and 2Laboratoire de Biologie des Tumeurs Humaines, Institut Gustave Roussy, Villejuif, France Latent membrane protein 2A (LMP-2A) is expressed in Epstein-Barr virus transformed B lymphocytes in vitro and has been detected in various types of EBV- associated malignancies. LMP-2A interferes with mem- brane signal transduction through phosphorylation of its hydrophilic N-terminal domain and binding of the cellular tyrosine kinases encoded byfvn and lyn. In vitro, the domain can block calcium influx and participate in signal transduction inducing cytokine production. These two activities are differently affected by site-directed mutagenesis of potentially phosphorylated amino acid residues. Several potential tyrosine protein kinase rec- ognition motifs have been identified including an antigen recognition motif (ARAM). ARAMs are activated by tyrosine phosphorylation that enables binding of tyro- sine protein kinases such as lyn and fvn. To assess the importance of potential sequence variation in natural EBV infection and in tumourigenesis, the sequence of the LMP-2A N-terminal domain was determined in 28 EBV isolates, including 14 fresh tumour isolates. Comparison of the corresponding sequences with the prototype B95 strain indicates that LMP-2 is generally conserved with a few base pair changes resulting in conservative amino acid changes in an occasional isolate. However, five single-base loci were frequently mutated, resulting in three patterns of sequence poly- morphism in exon 1 of LMP-2A. The patterns did not segregate with EBV Type 1 or Type 2 and were detected in both lymphoid and epithelial tissues. Four of the most frequent mutations at loci 166627, 166750, 166796 and 166805 (codons 23, 63, 79 and 82) could potentially affect tyrosine protein kinase binding motifs. The pivotal tyrosines (codons 74 and 85) and leucines (codons 77 and 88) of the LMP-2 ARAM were not affected in any of the isolates, suggesting that ARAM function is important for EBV infection in vivo. However, the inter- spacing positions 79 and 82 were distinct in more than 50% of the isolates. These prevalent polymorphisms could influence interaction of the LMP-2 cytoplasmic domain with specific cellular ligand proteins. Epstein-Barr virus (EBV) encodes two integral mem- brane proteins, latent membrane proteins (LMPs) 1 and 2 (Kieff & Liebowitz, 1990). There are two forms of LMP-2, LMP-2A and -2B, that are encoded by two transcripts which are spliced across the terminal repeats of the EBV DNA (Laux et al., 1988; Sample et al., 1989). Therefore, their transcription requires episomal forms of the viral genome or rare integrative events (Hurley et al., 1991). Each transcript results from the alternative splicing of a unique 5' exon 1, located at the right-end of the genome, while exons 2 to 9, located at the left-end of the genome, are common to both species. The LMP-2A 5' first exon (368 bp) encodes an N-terminal hydrophilic, intracytoplasmic domain (119 aa) while the LMP-2B first exon (154bp) is non-coding. The eight exons common to LMP-2A and -2B encode a highly hydro- * Author for correspondence. Fax +19199663015. e-mail nrt@med.unc.edu phobic transmembrane domain (1140 coding nucleo- tides; 378 aa) (Kieff & Liebowitz, 1990; Rabson et al., 1982). The LMP-2A and -2B proteins are known to be expressed in lymphoblastoid and Burkitt's lymphoma cell lines (Frech et al., 1990; Longnecker & Kieff, 1990). The LMP-2 genes are consistently transcribed in naso- pharyngeal carcinomas (NPC) and LMP-2A transcripts have also been detected in EBV-positive uncultured peripheral blood lymphocytes (Brooks et al., 1992; Busson et al., 1992; Qu & Rowe, 1992). The sequence of two overlapping cDNAs representative of the entire LMP-2A coding sequence was determined in C15, an NPC established in nude mice (Busson et al., 1988, 1992). When compared with the B95 sequence, exons 2 to 9 were highly conserved; on a segment of 1140 bp, five mutations were detected with only one resulting in a significant change, at codon 126, with the replacement of a leucine by a proline. In contrast, nine mutations were 0001-2626 © 1995SGM