Deletion of DNA encoding the first five transmembrane domains of Epstein-Barr virus latent membrane proteins 2A and 2B.

A recombinant Epstein-Barr virus (EBV) was constructed, with a positive-selection marker inserted at the site of a deletion of a DNA segment which encodes the first five transmembrane domains of LMP2A and LMP2B. Despite the mutation, the mutant recombinant EBV was able to initiate and maintain primary B-lymphocyte growth transformation in vitro. Cells transformed with the mutant recombinant were not different from wild-type virus transformants in initial or long-term outgrowth, sensitivity to limiting cell dilution, or serum requirement. Expression of EBNA1, EBNA2, EBNA3A, EBNA3C, and LMP1 and permissivity for lytic EBV infection were also unaffected by the LMP2 deletion mutation. These results complete the molecular genetic studies proving LMP2 is dispensable for primary B-lymphocyte growth transformation, latent infection, and lytic virus replication in vitro.     

PY motifs of Epstein-Barr virus LMP2A regulate protein stability and phosphorylation of LMP2A-associated proteins

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A amino-terminal domain. In this study, LMP2A PY motif mutant viruses were constructed to investigate the role of the LMP2A PY motifs. AIP4 was found to specifically associate with the LMP2A PY motifs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our original observation to EBV-infected cells. Mutation of both of the LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa protein. In addition, there was a modest increase in the constitutive phosphorylation of Syk and an unidentified 60-kDa protein. These results indicate that the PY motifs contained within LMP2A are important in regulating phosphorylation in EBV-infected LCLs, likely through the regulation of Lyn activity by specifically targeting the degradation of Lyn by ubiquination by Nedd4 family E3s. Despite differences between PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still exhibited a block in B-cell receptor (BCR) signal transduction as measured by the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation. EBV-transformed LCLs with mutations in the PY motifs were not different from wild-type LCLs in serum-dependent cell growth. Protein stability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.     

Epstein-Barr Virus nuclear protein EBNA3A is critical for maintaining lymphoblastoid cell line growth

To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.     

Latent Membrane Protein LMP2A Impairs Recognition of EBV-Infected Cells by CD8+ T Cells

The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.     

Identification of latent membrane protein 2A (LMP2A) domains essential for the LMP2A dominant-negative effect on B-lymphocyte surface immunoglobulin signal transduction.

Epstein-Barr virus (EBV) recombinants which carry three different deletion mutations in the LMP2A cytoplasmic amino-terminal domain were constructed. The presence of each mutation, LMP2A delta 21-36, LMP2A delta 21-64, and LMP2A delta 21-85, in EBV-infected transformed lymphoblastoid cell lines was confirmed by PCR analysis and Southern blot hybridization. Confirmation of mutant LMP2A protein expression was by immunofluorescence and immunoblotting with a newly identified rat monoclonal antibody that recognizes each of the LMP2A deletion mutations. Lymphoblastoid cell lines infected with recombinant EBV DNAs containing the mutations were analyzed for loss of LMP2A's dominant-negative effect on surface immunoglobulin signal transduction by monitoring induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication following surface immunoglobulin cross-linking. Domains of LMP2A important for induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication were identified.     

The last seven transmembrane and carboxy-terminal cytoplasmic domains of Epstein-Barr virus latent membrane protein 2 (LMP2) are dispensable for lymphocyte infection and growth transformation in vitro.

Specifically mutated Epstein-Barr virus (EBV) recombinants which truncate latent membrane protein 2A (LMP2A) and LMP2B after 260 of 497 amino acids and after 141 of 378 amino acids, respectively, were constructed. Despite truncation before the last seven transmembrane domains and the carboxy terminus, the mutant recombinants were not altered in initiation of primary B-lymphocyte infection or growth transformation, in expression of nuclear protein 1 or 2 or LMP1, or in induction of lytic EBV replication. Cells transformed by mutant virus recombinants were not different from wild-type virus transformants in initial or long-term outgrowth, sensitivity to limiting cell dilution, serum requirement, or clonogenic growth in soft agar. Together with similar analyses of a mutation stopping translation of the LMP2A amino-terminal cytoplasmic domain, these results indicate that LMP2 is not required for primary B-lymphocyte infection in vitro.     

Epstein-Barr病毒特异性T细胞对重组痘苗病毒表达LMP和EBNA2的靶细胞的识别

本文用EB病毒转化自体淋巴细胞所建立的类淋巴母细胞系(LCL),以及用EB病毒潜伏感染膜蛋白(LMP)基因和核蛋白-2(EBNA2)基因与痘苗病毒重组的重组病毒(Vac-LMP和Vac-EBNA2)感染的自身纤维母细胞,同时作为刺激细胞和靶细胞,以~(51)Cr释放法检测5例血清中EB病毒VCA—IgA抗体阳性者及1例阴性健康者外周血单个核细胞(PBMC)的特异性T细胞杀伤效应。结果表明,用自身LCL激活的EB病毒特异性T细胞杀伤效应高峰出现在第14～28天;参与杀伤性细胞免疫反应的T细胞亚群主要是T3、T8阳性的细胞毒性T细胞,其对靶细胞的识别及杀伤受HLA-I的限制。用重组牛痘病毒感染的纤维母细胞作靶细胞或刺激细胞,有1例供者可接受LMP,另1例可接受EBNA2的刺激,并对相应的靶细胞产生特异性T细胞杀伤反应,表明EB病毒-LMP和EBNA2可能既是EB病毒特异性T细胞的刺激抗原,又是其识别的靶抗原。     

An Epstein-Barr virus that expresses only the first 231 LMP1 amino acids efficiently initiates primary B-lymphocyte growth transformation

An Epstein-Barr virus (EBV) recombinant (MS231) that expresses the first 231 amino acids (aa) of LMP1 and is truncated 155 aa before the carboxyl terminus transformed resting B lymphocytes into lymphoblastoid cell lines (LCLs) only when the infected cells were grown on fibroblast feeder cells (K. M. Kaye et al., J. Virol. 69:675-683, 1995). Higher-titer MS231 virus has now been compared to wild-type (WT) EBV recombinants for the ability to cause resting primary B-lymphocyte transformation. Unexpectedly, MS231 is as potent as WT EBV recombinants in causing infected B lymphocytes to proliferate in culture for up to 5 weeks. When more than one transforming event is initiated in a microwell, the MS231 recombinant supports efficient long-term LCL outgrowth and fibroblast feeder cells are not required. However, with limited virus input, MS231-infected cells differed in their growth from WT virus-infected cells as early as 6 weeks after infection. In contrast to WT virus-infected cells, most MS231-infected cells could not be grown into long-term LCLs. Thus, the LMP1 amino-terminal 231 aa are sufficient for initial growth transformation but the carboxyl-terminal 155 aa are necessary for efficient long-term outgrowth. Despite the absence of the carboxyl-terminal 155 aa, MS231- and WT-transformed LCLs are similar in latent EBV gene expression, in ICAM-1 and CD23 expression, and in NF-kappaB and c-jun N-terminal kinase activation. MS231 recombinant-infected LCLs, however, require 16- to 64-fold higher cell density than WT-infected LCLs for regrowth after limiting dilution. These data indicate that the LMP1 carboxyl-terminal 155 aa are important for growth at lower cell density and appear to reduce dependence on paracrine growth factors.     

The Epstein-Barr virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte transformation; fibroblast cocultivation complements a critical function within the terminal 155 residues.

Recombinant Epstein-Barr viruses (EBVs) were made with mutated latent membrane protein 1 (LMP1) genes that express only the LMP1 amino-terminal cytoplasmic and six transmembrane domains (MS187) or these domains and the first 44 amino acids of the 200-residue LMP1 carboxy-terminal domain (MS231). After infection of primary B lymphocytes with virus stocks having small numbers of recombinant virus and large numbers of P3HR-1 EBV which is transformation defective but wild type (WT) for LMP1, all lymphoblastoid cell lines (LCLs) that had MS187 or MS231 LMP1 also had WT LMP1 provided by the coinfecting P3HR-1 EBV. Lytic virus infection was induced in these coinfected LCLs, and primary B lymphocytes were infected. In over 200 second-generation LCLs, MS187 LMP1 was never present without WT LMP1. Screening of over 600 LCLs infected with virus from MS231 recombinant virus-infected LCLs identified two LCLs which were infected with an MS231 recombinant without WT LMP1. The MS231 recombinant virus could growth transform primary B lymphocytes when cells were grown on fibroblast feeders. Even after 6 months on fibroblast feeder layers, cells transformed by the MS231 recombinant virus died when transferred to medium without fibroblast feeder cells. These data indicate that the LMP1 carboxy terminus is essential for WT growth-transforming activity. The first 44 amino acids of the carboxy-terminal cytoplasmic domain probably include an essential effector of cell growth transformation, while a deletion of the rest of LMP1 can be complemented by growth on fibroblast feeder layers. LMP1 residues 232 to 386 therefore provide a growth factor-like effect for the transformation of B lymphocytes. This effect may be indicative of the broader role of LMP1 in cell growth transformation.     

Epstein-Barr Virus Promotes Epithelial Cell Growth in the Absence of EBNA2 and LMP1 Expression

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10⁶ cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs.

Transformation-competent, replication-defective Epstein-Barr virus (EBV) recombinants which are deleted for 18 kbp of DNA encoding the largest EBNA intron and for 58 kbp of DNA between the EBNA1 and LMP1 genes were constructed. These recombinants were made by transfecting three overlapping cosmid-cloned EBV DNA fragments into cells infected with a lytic replication-competent but transformation-defective EBV (P3HR-1 strain) and were identified by clonal transformation of primary B lymphocytes into lymphoblastoid cell lines. One-third of the lymphoblastoid cell lines were infected with recombinants which had both deletions and carried the EBNA2 and EBNA3 genes from the transfected EBV DNA and therefore are composed mostly or entirely from the transfected EBV DNA fragments. The deleted DNA is absent from cells infected with most of these recombinants, as demonstrated by Southern blot and sensitive PCR analyses for eight different sites within the deleted regions. Cell growth and EBNA, LMP, and BZLF1 gene expression in lymphoblastoid cell lines infected with these recombinants are similar to those in cells infected with wild-type EBV recombinants. Together with previous data, these experiments reduce the complexity of the EBV DNA necessary for transformation of primary B lymphocytes to 64 kbp. The approach should be useful for molecular genetic analyses of transforming EBV genes or for the insertion of heterologous fragments into transforming EBV genomes.     

Autocrine CCL3 and CCL4 Induced by the Oncoprotein LMP1 Promote Epstein-Barr Virus-Triggered B Cell Proliferation

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.     

A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1.

Recent cDNA cloning and sequencing of two Epstein-Barr virus (EBV)-specific mRNAs from latently infected cultures revealed that these RNAs are encoded across the fused terminal repeats of the viral genome and that they are likely to encode two nearly identical proteins with the same transmembrane domains. The smaller predicted protein (LMP2B) lacks 119 amino-terminal amino acids found in the larger one (LMP2A). To test whether these proteins are expressed in latently infected lymphocytes, antibodies to the LMP2 proteins were derived by immunizing rabbits with TrpE-LMP2A fusion proteins. Affinity-purified LMP2-specific antibodies recognized 54- and 40-kilodalton proteins, corresponding to LMP2A and LMP2B, in immunoblots of rodent fibroblasts stably transfected with eucaryotic expression plasmids containing either the LMP2A or LMP2B cDNA. Similar-size proteins were also identified in immunoblots of latently infected lymphocytes. LMP2A localized to membranes in cellular fractionation studies. In immunofluorescent studies, LMP2 localized in the plasma membrane of EBV-infected lymphocytes, with the majority of reactivity confined to the region of the LMP1 patch. This reactivity was detected in almost all lymphoblastoid cells latently infected with EBV.     

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.