The only domain which distinguishes Epstein-Barr virus latent membrane protein 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro; LMP2A is therefore nonessential

Abstract

Using second-site homologous recombination, Epstein-Barr virus (EBV) recombinants were constructed which carry an LMP2A mutation terminating translation at codon 19. Despite the absence of LMP2A or LMP2A cross-reactive protein, the recombinants were able to initiate and maintain primary B-lymphocyte growth transformation in vitro. EBNA1, EBNA2, and LMP1 expression was unaffected by the LMP2A mutation. The LMP2A mutant recombinant EBV-infected lymphoblastoid cell lines (LCLs) were identical to wild-type recombinant EBV-infected control LCLs with respect to initial outgrowth, subsequent growth, sensitivity to limiting cell dilution, sensitivity to low serum, and growth in soft agarose. The permissivity of LCLs for lytic EBV infection and virus replication was also unaffected by the LMP2A mutation.

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo

“…A control reaction with pgp110HYG as the template also yielded the 207-bp product. As expected, no amplified product was detected with these primers in LCL2, a B95-8 EBV-transformed lymphoblastoid cell line (19), M12, a murine B-lymphocyte line (15), BJAB, and EBV-negative Burkitt's lymphoma cell line (23), or in reaction mixtures containing primers only ( Fig. 2A).…”

“…3B, line 2) was detected with the BamHI A probe (Fig. 3A, lanes 7,19,21). In LCLs infected only with B95-110HYG, this probe detected a 13,617-bp fragment (Fig.…”

“…A second plasmid, pSVNaeZ, was cotransfected to express the Zta EBV immediate-early transactivator and induce lytic infection in the B95-8 cells (41). To obtain lymphoblastoid cell lines (LCLs) infected with recombinant EBV gp110 mutants, primary B lymphocytes were infected with cellfree virus prepared from the transfected B95-8 cells, plated in 96-well microtiter dishes, and selected with hygromycin B (50 g/ml), all as previously described (19,21,22). Multiple wells of hygromycin-resistant LCLs were generated from two independent experiments; nine of these were expanded and studied further.…”

“…pSG5 DNA (25 g) was transfected in experiments with the vector alone. Prior to all analyses, the cells were cultured for an additional 3 to 5 days in media containing 20 ng of tetradecanoyl phorbol acetate per ml to further induce lytic replication (19,20).…”

5' Coding and regulatory region sequence divergence with conserved function of the Epstein-Barr virus LMP2A homolog in herpesvirus papio

“…Genetic recombinant analysis indicates that LMP2A is not essential for EBV replication or immortalization of B cells in vitro (31,(44)(45)(46). However, LMP2A is expressed in vivo by latently infected B cells circulating in the peripheral blood and in nasopharyngeal carcinomas (4,7,55).…”