Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, N15 W7, Kita-ku, 060-8638 Sapporo, Japan1
Second Department of Surgery, Hokkaido University School of Medicine, Sapporo, Japan2
Author for correspondence: Kenzo Takada. Fax +81 11 717 1128. e-mail kentaka{at}med.hokudai.ac.jp
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Abstract |
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Main text |
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LMP2A and LMP2B are transcribed from unique and common exons (Laux et al., 1988 ; Sample et al., 1989
). The first exon is unique to LMP2A and encodes a 119 amino acid cytoplasmic amino-terminal domain. The remaining eight exons are common with LMP2B, and encode 12 hydrophobic membrane-spanning domains and a 27 amino acid cytoplasmic carboxy-terminal domain. The amino-terminal part of LMP2A is associated with src family protein tyrosine kinases (Burkhardt et al., 1992
; Longnecker et al., 1991
). LMP2A is a substrate for these protein kinases, acts as a dominant negative regulator of lyn and fyn protein kinase activities, and interferes with signal transduction after cross-linking of cell surface immunoglobulin (sIg) (Miller et al., 1993
, 1994a
, 1995
). Cross-linking of sIg efficiently activates a switch to lytic EBV gene expression in latently EBV-infected cells (Takada, 1984
; Takada & Ono, 1989
). LMP2A prevents lytic reactivation in response to sIg cross-linking in EBV-immortalized B-lymphocytes (Miller et al., 1994b
), and is thought to maintain a latent state of EBV infection in vivo, because only EBNA1 and LMP2A were reported to be expressed in latently EBV-infected B-cells in vivo (Chen et al., 1995
; Miyashita et al., 1997
; Qu & Rowe, 1992
; Tierney et al., 1994
).
Concerning the role of LMP2A in B-cell immortalization, there have been reports from two groups. R. Longnecker and colleagues reported that LMP2A was dispensable for B-cell immortalization. Two EBV mutants were generated: one a knockout of the first exon of the LMP2A gene (Longnecker et al., 1992 ; Speck et al., 1999
), and the other of the last seven transmembrane and carboxy-terminal cytoplasmic domains (Longnecker et al., 1993
), which are shared with LMP2B, and therefore the latter mutant is deficient in both LMP2A and LMP2B genes. An EBV recombinant was generated in EBV-producing P3HR-1 cells and B95-8 cells as a mixture of wild EBV and mutant EBV, which was then used to immortalize primary B-lymphocytes. LMP2A- or LMP2A/2B-knockout EBV released from immortalized B-cells was demonstrated to have an ability to immortalize primary B-lymphocytes. However, the assays were not quantitative because the virus preparations always contained both wild and mutant EBV. On the other hand, W. Hammerschmidt and colleagues (Brielmeier et al., 1996
) reported that LMP2 was important for efficient B-cell immortalization. Knockout EBV was generated in an E. coli F-factor-based plasmid and packaged in P3HR-1 cells, which were infected with EBNA2-deleted, transformation-incompetent EBV. This assay, however, could not evaluate the function of LMP2A, because the knocked-out LMP2 regions were common to both LMP2A and LMP2B.
Our present study aimed to quantitatively determine the role of LMP2A in B-cell immortalization by using a pure recombinant EBV preparation generated by an Akata cell system (Shimizu et al., 1996 ). We also studied whether the small amount of LMP2A expressed in Akata cells, which must be similar to that in latently EBV-infected B cells in vivo, had any effect on preventing activation of latent EBV.
The Akata cell line, derived from Burkitts lymphoma, was originally 100% EBV-positive (Takada et al., 1991 ). We isolated EBV-positive and -negative subclones from the parental Akata cell culture by the limiting dilution method. EBV-positive Akata cells (Akata+ cells) have
20 copies of EBV plasmid per cell. The first exon of the LMP2A gene of an EBV plasmid was disrupted by insertion of the neomycin resistance gene (neor) by homologous recombination (Fig. 1A
). The targeting plasmid was the BglII fragment encompassing the first exon of LMP2A (Sample et al., 1989
), disrupted by insertion of neor. The plasmid was transfected into Akata+ cells by the electroporation method. Transfected Akata cells were cultured in 96-well, flat-bottom plates at 5000 cells per well in complete culture medium containing 700 µg/ml of G418 (Life Technologies).
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Then, EBV-negative Akata cells (Akata-, 5x106) (Shimizu et al., 1994 ) were infected with the virus preparation from G418-resistant Akata cells, which contained a mixture of wild-type EBV and recombined EBV. Two days after infection, cells were plated in 96-well, flat-bottom plates at 5000 cells per well with complete medium containing 700 µg G418/ml. Twelve G418-resistant clones were examined and proved to be infected with recombinant EBV only. Southern blot analysis indicated that the recombinant EBV-infected Akata- cells had the neor DNA exactly recombined into the first exon of the LMP2A gene (Fig. 1B
, C
). RTPCR analysis indicated that LMP2A-knockout EBV-infected Akata cells were negative for LMP2A mRNA expression (Fig. 1D
). Furthermore, immunoblot analysis indicated that LCLs immortalized by LMP2A-knockout EBV were negative for LMP2A expression (Fig. 1E
).
At first, we compared virus production by Akata cells harbouring the LMP2A-knockout EBV with that of Akata cells harbouring recombinant EBV with insertion of the neor gene at the BXLF1 gene (referred to as wild EBV), which is known to be non-essential for EBV infection and replication. In three cell clones, LMP2A-knockout EBV-infected Akata cells produced 4·2, 4·5 and 5·3 µg of viral DNA per litre of culture supernatant, and wild EBV-infected Akata cells produced 2·9, 4·0 and 5·8 µg of viral DNA per litre of culture. Equal amounts of virus (corresponding to 4 ng of viral DNA) from both wild-type EBV and LMP2A-knockout EBV preparations were diluted 10-fold, inoculated to cord blood mononuclear cells (106 cells), and their ability to immortalize primary B-lymphocytes was assessed. In two separate experiments, the 50% immortalization doses per 4 ng of viral DNA were 104·8 and 105·2 for LMP2A-knockout EBV, and 105·2 and 104·6 for wild EBV. These results indicated that there was no difference in immortalization efficiency between wild EBV and LMP2A-knockout EBV.
Akata cells express a small amount of LMP2A that is detectable by RTPCR but not by immunoblot analysis. We examined whether this amount of LMP2A expression had any effect on sIg-mediated calcium mobilization. Wild EBV-infected and LMP2A-knockout EBV-infected Akata cells were treated with anti-Ig, and their intracellular free calcium levels were measured by flow cytometry (Table 1). Both wild EBV-infected and LMP2A-knockout EBV-infected Akata cells exhibited similar changes in intracellular calcium.
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Next we examined whether high-level expression of LMP2A comparable to the level in LCL could inhibit calcium mobilization induced by anti-Ig treatment. The LMP2A expression plasmid was generated from cDNA of Akata EBV-immortalized LCL by a PCR-based strategy. Total cellular RNA was reverse-transcribed using the 3LMP2A primer (GCACATTGGGTTTATTGTAGTGTTTGTAAATAC). The cDNA was then subjected to 35 cycles of PCR using primers 3LMP2A and 5LMP2A (TTAGCGCTGCTGCAGCTATGGGGTCCCTAG). The amplified DNA was sequenced to confirm the absence of any mutations introduced by PCR. Compared with the nucleotide sequence of the B95-8 strain (Baer et al., 1984 ), there were three base changes, which resulted in three amino acid changes (tyrosine-23 to aspartate, and serine-348 and -444 to isoleucine). Tyrosine-23 has been reported to be dispensable for blocking B-cell signal transduction (Fruehling et al., 1996
). The cDNA of LMP2A was further cloned into the pSG5 vector containing neor and transfected into BJAB, Akata- and Akata+ cells. Cell clones that had LMP2A levels similar to those of LCL were isolated (Fig. 2A
, B
, C
) and examined for their calcium mobilization response to anti-Ig treatment. In all three cell lines, LMP2A-transfected cell clones showed reduced numbers of cells responding, as well as a reduction in the degree to which responding cells mobilized calcium, compared with those of vector-transfected clones (Table 1
).
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We then compared the level of lytic antigen expression after anti-Ig treatment between wild EBV-infected and LMP2A-knockout EBV-infected LCL. The results indicated that wild EBV-infected LCLs showed very little or no induction of EBV lytic antigens, while LMP2A-knockout EBV-infected LCLs produced EBV early antigens (EA) and viral capsid antigens (VCA) in 5% and 1% of cells, respectively, after anti-Ig treatment.
Next Akata+ cells, which expressed a small amount of LMP2A, were transfected with the LMP2A plasmid, and cell clones that constitutively expressed LMP2A at a level similar to LCL were isolated. These cell clones became less permissive for virus replication as compared with Akata+ cell clones transfected with the neor plasmid as a control.
Our results showed clearly that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in peripheral lymphocytes. Brielmeier et al. (1996) reported that the deletion of both LMP2A and LMP2B genes from EBV lowered the lymphocyte immortalization efficiency remarkably. It remains to be studied whether LMP2B plays some role in lymphocyte immortalization.
LCL express a high level of LMP2A protein, which can be detected by Western blot. The high-level expression of LMP2A in LCL is caused by transactivation of the EBNA2 protein (Zimber et al., 1993 ). On the other hand, Akata cells are negative for EBNA2 expression and express little LMP2A. The analysis of peripheral blood lymphocytes by PCR showed that only EBNA1 and LMP2A were expressed in EBV latency in vivo (Chen et al., 1995
; Miyashita et al., 1997
; Qu & Rowe, 1992
; Tierney et al., 1994
). Although the level of LMP2A expression in peripheral lymphocytes has not been measured quantitatively, the absence of EBNA2 expression suggests a low level of LMP2A expression in these cells. Our results showed that the low level of LMP2A expressed in Akata cells could not block the EBV activation by cross-linking of sIg, suggesting that a low level of LMP2A expression in in vivo latency could not block EBV activation.
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Acknowledgments |
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References |
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Received 29 November 2000;
accepted 1 February 2001.