Latent Membrane Protein 1 Sequence Research Articles

Evolutionary analysis of LMP-1 genetic diversity in EBV-associated nasopharyngeal carcinoma: Bioinformatic insights into oncogenic potential

EBV latent membrane protein 1 (LMP-1) is an important oncogene involved in the induction and maintenance of EBV infection and the activation of several cell survival and proliferative pathways. The genetic diversity of LMP-1 has an important role in immunogenicity and tumorigenicity allowing escape from host cell immunity and more metastatic potential of LMP-1 variants. This study explored the evolutionary of LMP-1 in EBV-infected patients at an advanced stage of nasopharyngeal carcinoma (NPC). Detection of genetic variability in LMP-1 genes was carried out using Sanger sequencing. Bioinformatic analysis was conducted for translation and nucleotide alignment. Phylogenetic analysis was used to construct a Bayesian tree for a deeper understanding of the genetic relationships, evolutionary connections, and variations between sequences. Genetic characterization of LMP-1 in NPC patients revealed the detection of polymorphism in LMP-1 Sequences. Motifs were identified within three critical LMP-1 domains, such as PQQAT within CTAR1 and YYD within CTAR2. The presence of the JACK3 region at specific sites within CTAR3, as well as repeat regions at positions (122−132) and (133–143) within CTAR3, was also annotated. Additionally, several mutations were detected including 30 and 69 bp deletions, 33 bp repeats, and 15 bp insertion. Although LMP-1 strains appear to be genetically diverse, they are closely related to 3 reference strains: prototype B95.8, Med- 30 bp deletion, and Med + 30 bp deletion. In our study, one of the strains harboring the 30 bp deletion had both bone and bone marrow metastasis which could be attributed to the fact that LMP-1 is involved in tumor metastasis, evasion and migration of NPC cells. This study provided valuable insights into genetic variability in LMP-1 sequences of EBV in NPC patients. Further functional studies would provide a more comprehensive understanding of the molecular characteristics, epidemiology, and clinical implications of LMP-1 polymorphisms in EBV-related malignancies.

New Insights from Elucidating the Role of LMP1 in Nasopharyngeal Carcinoma.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV) oncogenic protein that has no intrinsic enzymatic activity or sequence homology to cellular or viral proteins. The oncogenic potential of LMP1 has been ascribed to pleiotropic signaling properties initiated through protein-protein interactions in cytosolic membrane compartments, but the effects of LMP1 extend to nuclear and extracellular processes. Although LMP1 is one of the latent genes required for EBV-immortalization of B cells, the biology of LMP1 in the pathogenesis of the epithelial cancer nasopharyngeal carcinoma (NPC) is more complex. NPC is prevalent in specific regions of the world with high incidence in southeast China. The epidemiology and time interval from seroconversion to NPC onset in adults would suggest the involvement of multiple risk factors that complement the establishment of a latent and persistent EBV infection. The contribution of LMP1 to EBV pathogenesis in polarized epithelia has only recently begun to be elucidated. Furthermore, the LMP1 gene has emerged as one of the most divergent sequences in the EBV genome. This review will discuss the significance of recent advances in NPC research from elucidating LMP1 function in epithelial cells and lessons that could be learned from mining LMP1 sequence diversity.

Clonal deleted latent membrane protein 1 variants of Epstein-Barr virus are predominant in European extranodal NK/T lymphomas and disappear during successful treatment.

Extranodal natural killer/T-cell lymphomas (NK/TL), rare in Europe, are Epstein-Barr virus (EBV) associated lymphomas with poor outcomes. Here, we determined the virus type and analyzed the EBV latent membrane protein-1 (LMP1) gene sequence in NK/TL from French patients. Six clones of viral LMP1 were sequenced by Sanger technology in blood from 13 patients before treatment with an l-asparaginase based regimen and, for 8 of them, throughout the treatment. Blood LMP1 sequences from 21 patients without any known malignancy were tested as controls. EBV Type A was identified for 11/13 patients and for all controls. Before treatment, a clonal LMP1 gene containing a 30 bp deletion (del30) was found in 46.1% of NK/TL and only in 4.8% of controls. Treatment was less effective in these patients who died more rapidly than the others. Patients with a deleted strain evolving toward a wild-type strain during treatment reached complete remission. The LMP1 gene was sequenced by highly sensitive next-generation sequencing technology in five NK/TL nasopharyngeal biopsies, two of them originating from the previous patients. Del30 was present in 100% of the biopsies; two viruses at least coexisted in three biopsies. These results suggest that del30 may be associated with poor prognosis NK/TL and that strain evolution could be used as a potential marker to monitor treatment.

Genetic Diversity of EBV-Encoded LMP1 in the Swiss HIV Cohort Study and Implication for NF-Κb Activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Epstein–Barr virus strains defined by the latent membrane protein 1 sequence characterize Thai ethnic groups

Epstein-Barr virus (EBV) is ubiquitous in the human population and seroepidemiological studies have revealed that more than 90% of adults are infected with the virus in Thailand. It has been suggested that latent membrane protein 1 (LMP1) variants may differ in their tumorigenicity and geographical localization. The distribution of LMP1 variants of EBV in the Thai population was studied. A total of 259 LMP1 sequences from ten Thai ethnic groups (Lahu, Lisu, Shan, Red Karen, White Karen, Hmong, Akha, Mlabri, Moken and Urak Lawoi) were studied using direct PCR sequencing. Nucleotide sequences corresponding to the C terminus of the LMP1, including previously published sequences from central and southern Thais, were used in the phylogenetic analysis. Five strains--the B95-8 prototype, China 1, China 2, Mediterranean (Med) and SEA 2--were identified in ethnic groups in Thailand. The major strain and the distribution pattern differed by group and location. When the ethnic groups were classified by linguistic group, the prevalence of the SEA 2 strain was significantly different between Austro-Thais and other linguistic groups (P=0.0001), whereas, among Tibeto-Burman linguistic groups, the prevalence of the Med strain was different between matrilocal and patrilocal groups (P=0.0245). The distribution of LMP1 strains in ethnic minorities in Thailand is associated with ethnogeographical factors and the social/marriage system. This study thus provides evidence for the importance of interactions between populations in virus diversity.

Epstein-Barr Virus Associated Modulation of Wnt Pathway Is Not Dependent on Latent Membrane Protein-1

Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, β-catenin, Glycogen Synthase Kinase 3ß (GSK3β), axin and α-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and β-catenin interaction and did not induce transcriptional activation of β-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.

The vIL-10 gene of the Epstein-Barr virus (EBV) is conserved in a stable manner except for a few point mutations in various EBV isolates

A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame 1 (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vIL-10) gene was analyzed using PCR-direct sequencing in African Burkitt's lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had China1-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.

Epstein?Barr virus genotypes in multiple sclerosis

Multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection, but the relationship between the virus and the disease is not clear. As many different types of EBV exist, it is possible that MS is caused by one particular type of EBV. Objectives - The aim of this study was to determine whether MS is associated with a particular genotype of EBV. We collected blood from MS patients and controls, amplified and sequenced the latent membrane protein-1 (LMP-1) gene, and compared the groups. We found a variety of LMP-1 sequences in both MS and controls, with no significant differences between the groups. Conclusion - We conclude that MS is not associated with a particular genotype of EBV.

Molecular analysis of the Epstein-Barr virus LMP1 in Hodgkin lymphoma from Argentina and Brazil: Identification of a distinct LMP1 deleted variant

21099 Background: Epstein Barr virus (EBV), a human oncogenic virus, has two types, EBV1 and 2, and several polymorphic markers allow individual variants to be distinguished. Variations in EBV latent membrane protein 1 (LMP1) gene could define a more oncogenic strain or reflect geographic EBV origin. By sequence analysis of C-ter, N-ter and promoter (prom) regions, 4 distinct EBV groups (A to D) have been defined. Aim: To analyze LMP1 molecular variability of two different groups of Hodgkin`s lymphoma (HL) from the same geographic area. Methods: EBV association by EBERs in situ hibridization and LMP1 immunohistochemistry was analyzed in 27 HL from Argentina (Arg) and 36 HL from Brazil (Br). EBV type was assessed by EBNA PCR. C-ter LMP1 PCR was done in all cases. Sequencing of C-ter, N-ter and prom regions was done in 16 patients. Results: 21/27 (78%) Arg HL and 31/36 (86%) Br HL were EBV1, while 6/27 (22%) and 4/36 (11%) were EBV2, respectively (p= 0.29). Coinfection was observed in 1 (3%) Br HL. LMP1 deleted (del) variant was observed in 20/27 (74%) Arg HL and 20/36 (55%) Br HL (p= 0.18), as well as in non-neoplastic controls, 4/11 and 3/10 (36 and 30%) from Arg and Br respectively. Most LMP1 del displayed high number (5–7) of 33bp repeats (86% LMP1 del Br HL and 77% LMP1 del Arg HL) compared with LMP1 wt that exhibited low number (3–4) 33bp repeats (68% LMP1 wt Br HL and 100% LMP1 wt Arg HL). Analysis of LMP1 sequences showed that: LMP1 wt C-ter regions had unmutated N-ter and prom as B95.8, A and B groups (31%), except for a case showing new mutations. LMP1 del C-ter regions showed molecular identity with C group, but they showed new, undescribed mutations in prom and N-ter (63%). Conclusions: We found high frequency of LMP1 del variants in HL from Argentina and Brazil, which was associated with high number of 33bp repeats in C-ter region. This suggests a role for this variant in lymphomagenesis. A new molecular variant with characteristic promoter and N-ter mutations was identified in LMP1 del cases, which could be proposed as a regional South American variant. No significant financial relationships to disclose.

Analysis of LMP1 variants of EBV in Southern Thailand: Evidence for strain-associated T-cell tropism and pathogenicity

Background The latent membrane protein 1 (LMP1) of Epstein–Barr virus (EBV) has sequence heterogeneity. Some of the variants are associated with altered tumorigenic activity and show geographically specific localization. In Thailand, the EBV genome is frequently detected in circulating T cells of T-cell diseases. Objective To determine the role of EBV LMP1 variation in the genesis of T-cell diseases, we focused on virus factors and analyzed EBV strains in Thailand. Study design EBV DNA was extracted from 18 healthy individuals and 45 patients with T-cell diseases in Southern Thailand and 30 healthy individuals in Central Thailand. By using PCR-direct sequencing method, nucleotide sequences corresponding to the carboxyl terminus of the LMP1 were determined. Results Four known strains, B95-8 prototype, China 1, China 2 and Mediterranean (Med) and two novel strains, Southeast Asia 1 (SEA 1) and Southeast Asia 2 (SEA 2) were identified. The prevalence of China 2 strain was significantly different ( p = 0.006) between Central and Southern Thailand. Higher prevalence ( p = 0.026) of 30-bp deletion type in the Southern Thais was observed. The LMP1 Med strain was associated with the worse prognosis ( p = 0.029). Among T-cell diseases patients, CD3 +-cell oriented infection was recognized in SEA1 strain ( p = 0.025). Conclusion The distribution of EBV strains may be associated with geographic/ethnic and clinical background in the Thai population. Certain EBV strains defined by their LMP1 sequence may influence cell tropism, disease association, or disease severity.

High frequency of latent membrane protein‐1 30‐bp deletion variant with specific single mutations in Epstein‐Barr virus‐associated nasopharyngeal carcinoma in Moroccan patients

Latent membrane protein 1 (LMP-1) is an Epstein-Barr virus-encoded oncoprotein expressed in approximately 50-70% of nasopharyngeal carcinoma (NPC). Previous studies have shown that NPC-derived LMP-1 variants carrying 30 bp deletion and specific mutations in the 3'C-terminal region confer high oncogenic potential and a weak immunogenicity. Although numerous polymorphism studies of LMP-1 have been carried out so far in the Asian population with NPC, very little is known in this regard on NPC patients from Northern Africa where there is a significantly high occurrence of this tumor. In our study, we examined the frequency of different LMP-1 sequence variants derived from Moroccan NPC patients. As compared to healthy donors, NPC patients showed a high prevalence of the 30bp deletion variant of LMP-1 (i.e. 84% vs. 36%; p<0.0005). Moreover, the del-LMP-1 variant derived from NPC tumors shared identical amino acid substitutions at positions 322, 334, 338, 352 and 366 with the Mediterranean (Med) variant, whereas those derived from peripheral blood mononuclear cells (PBMC) had similar mutation pattern as China1 variant. Additional mutations within the 342-352 regions (identified in LMP-1 variants without deletion derived from NPC tumors) were not found in healthy donors' PBMC. Our results support the assumption that the distribution of LMP-1 variants in NPC tumors co-segregate with geographic regions. Indeed, Med variant is found more frequently in tumors from NPC Moroccan patients, whereas China1 variant is more prevalent in tumors from NPC patients in endemic regions for NPC.

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.

The molecular epidemiology and evolution of Epstein-Barr virus: sequence variation and genetic recombination in the latent membrane protein-1 gene.

The phylogeny and evolution of Epstein-Barr virus (EBV) genetic variation are poorly understood. EBV latent membrane protein-1 (LMP-1) gene sequences are especially heterogeneous and may be useful as a tool for EBV genotype identification. Therefore, LMP-1 sequences obtained directly from EBV-infected human tissues were examined by PCR amplification and cloning. EBV genotypes were defined as "strains" from among 22 identified LMP-1 sequence patterns. Three molecular mechanisms were identified by which genetic diversity arises in the LMP-1 gene: point mutation, sequence deletion or duplication, and homologous recombination. The rate of LMP-1 gene evolution was found to be accelerated by coinfection with multiple EBV strains. The results of this study refine our understanding of LMP-1 sequence variation and enable accurate discrimination between independent EBV infection events and the consequence of intrahost EBV evolution. Thus, this LMP-1 sequence-based approach to EBV molecular epidemiology will facilitate the study of intrahost EBV infection, coinfection, and persistence.

Sequence Analysis of the Epstein-Barr Virus (EBV) Latent Membrane Protein-1 Gene and Promoter Region: Identification of Four Variants Among Wild-Type EBV Isolates

Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European wild-type virus isolates, we sequenced the LMP1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D. The widespread prevalence of LMP-1 sequence variations, particularly the Xho I polymorphism and the 30-bp deletion, indicate that they cannot be used as simple markers for oncogenic viruses related to particular forms of EBV-associated tumor. Several of the structural changes detected occur, however, at sites where they may affect transcription, translation, or function of LMP-1. Future in vitro studies should aim to establish the functional importance of variations at these sites.

Sequence Analysis of the Epstein-Barr Virus (EBV) Latent Membrane Protein-1 Gene and Promoter Region: Identification of Four Variants Among Wild-Type EBV Isolates

AbstractSequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European wild-type virus isolates, we sequenced the LMP1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D. The widespread prevalence of LMP-1 sequence variations, particularly the Xho I polymorphism and the 30-bp deletion, indicate that they cannot be used as simple markers for oncogenic viruses related to particular forms of EBV-associated tumor. Several of the structural changes detected occur, however, at sites where they may affect transcription, translation, or function of LMP-1. Future in vitro studies should aim to establish the functional importance of variations at these sites.

Sequence variation in the Epstein-Barr virus latent membrane protein 1.

The sequence of the latent membrane protein 1 (LMP-1) gene was analysed in Epstein-Barr virus (EBV) isolates from specific regions representing both type 1 and type 2 EBV. A predominant strain marked by an XhoI restriction enzyme polymorphism (REP) within the LMP-1 gene has been identified in type 1 EBV in nasopharyngeal carcinoma (NPC) from Southern China. This polymorphism was also present in type 2 EBV in NPC from Alaska. In this study, the sequence of the LMP-1 gene was determined in these samples representing type 1 and type 2 EBV and was compared with the prototype lymphoid strains. Consistent nucleotide variation in the amino terminus of LMP-1 was identified in strains marked by the XhoI REP. These changes were present in both EBV type 1 and type 2 strains. Three types of sequence variation were detected in the carboxy terminus of LMP-1. The LMP-1 sequences differed in the number of an 11 amino acid repeat element. In the prototype EBV type 1 (B95-8) sequence and in the type 1 Raji and type 2 HR-1 strains, the third repeat element contained an insertion of 5 amino acids that were also the first five unique amino acids after the last partial repeat element. The third variation was a deletion of amino acids 343 to 352 of the B95-8 LMP-1. This deletion was detected in the type 1 Chinese EBV strains, but was not detected in the type 2 Alaskan strains although the Chinese and Alaskan strains have nearly identical amino acid changes at the amino terminus. Numerous other amino acid changes were detected in the carboxy terminus which did not cosegregate with either EBV type, amino acid changes in the amino terminus, or specific geographic regions. These data indicate that EBV strains can be distinguished by sequence differences within LMP-1 and that unlike the divergence between type 1 and type 2 EBV in Epstein-Barr nuclear antigen sequences, different EBV types are nearly identical in LMP-1 sequence.

Delineation of the cis-acting element mediating EBNA-2 transactivation of latent infection membrane protein expression.

To delineate the cis-acting element through which EBNA-2 transactivates latent membrane protein 1 (LMP1), we assayed the effect of EBNA-2 on the activity of LMP1 promoter upstream deletion mutants in the context of the LMP1 or heterologous promoters controlling chloramphenicol acetyltransferase (CAT) reporter gene expression in Epstein-Barr virus-negative Burkitt lymphoma cells. Assays of progressive 5' deletions of the LMP1 promoter revealed low constitutive and at least eightfold EBNA-2-stimulated activity from -512 to +40 (-512/+40), -334/+40, and -234/+40 LMP1CAT plasmids. More extensive 5'-deleted -205/+40, -155/+40, and -147/+40 LMP1CAT plasmids also had low constitutive activity but were not EBNA-2 responsive. The most 5'-deleted -55/+40 LMP1CAT plasmid had moderate constitutive activity and was not EBNA-2 inducible. Either orientation of the -334/+40 LMP1 sequence conferred EBNA-2 responsiveness when positioned upstream of an enhancerless simian virus 40 or herpes simplex virus thymidine kinase (TK) promoter. EBNA-2 and the cis-acting LMP1 DNA were both required to increase TK promoter-initiated mRNA, indicating that the EBNA-2 effect is at the transcriptional level. Further deletion analysis of the EBNA-2-responsive cis-acting element defined a -234/-92 LMP1 DNA fragment which conveyed EBNA-2 responsiveness to the herpes simplex virus TK promoter. The 5' 30 bp between -234 and -205 were essential for EBNA-2 responsiveness. Thus, these experiments define a 142-bp cis-acting element which is sufficient for conveying EBNA-2 responsiveness and an essential 30-bp component of that element. The role of this element in LMP1 and LMP2B expression and its possible role in LMP2A expression are discussed.