Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation.

Recombinant Epstein-Barr viruses (EBV) with a translation termination codon mutation inserted into the nuclear protein 3A (EBNA-3A) or 3C (EBNA-3C) open reading frame were generated by second-site homologous recombination. These mutant viruses were used to infect primary B lymphocytes to assess the requirement of EBNA-3A or -3C for growth transformation. The frequency of obtaining transformants infected with a wild-type EBNA-3A recombinant EBV was 10 to 15%. In contrast, the frequency of obtaining transformants infected with a mutant EBNA-3A recombinant EBV was only 1.4% (9 mutants in 627 transformants analyzed). Transformants infected with mutant EBNA-3A recombinant virus could be obtained only by coinfection with another transformation-defective EBV which provided wild-type EBNA-3A in trans. Cells infected with mutant EBNA-3A recombinant virus lost the EBNA-3A mutation with expansion of the culture. The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain the EBNA-3A mutation indicate that EBNA-3A is essential or critical for lymphocyte growth transformation and that the EBNA-3A mutation has a partial dominant negative effect. Five transformants infected with mutant EBNA-3C recombinant virus EBV were also identified and expanded. All five also required wild-type EBNA-3C in trans. Serial passage of the mutant recombinant virus into primary B lymphocytes resulted in transformants only when wild-type EBNA-3C was provided in trans by coinfection with a transformation-defective EBV carrying a wild-type EBNA-3C gene. A secondary recombinant virus in which the mutated EBNA-3C gene was replaced by wild-type EBNA-3C was able to transform B lymphocytes. Thus, EBNA-3C is also essential or critical for primary B-lymphocyte growth transformation.     

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.     

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.     

A selectable marker allows investigation of a nontransforming Epstein-Barr virus mutant.

The derivation of specifically mutated Epstein-Barr virus (EBV) recombinants is dependent on strategies to identify, enumerate, and clone infected B lymphocytes. In recent experiments, EBV recombinants containing a positive selection marker were identified and cloned in B-lymphoma (BL) cells infected and then plated under selective conditions (F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). We now use BL cells, for the first time, as hosts for assaying and cloning otherwise isogenic EBV recombinants carrying a hygromycin phosphotransferase (HYG) gene linked to either a nontransforming deletion mutant or a transforming wild-type EBV nuclear antigen 2 (EBNA-2) gene. Both types of recombinants converted BL cells to hygromycin resistance with similar efficiency, formed episomes, and usually expressed only EBNA-1. Only the wild-type EBNA-2 HYG gene EBV recombinant transformed primary B lymphocytes. This strategy of assaying virus on BL and primary B lymphocytes makes possible the direct assessment of the transforming efficiency of an EBV recombinant. The resultant infected BL cells are also useful for the characterization of the nontransforming recombinant EBV genomes. The HYG gene insertion in the BHLF1 open reading frame eliminated BHLF1 protein expression. The insertion and resulting BHLF1 mutation did not interfere with primary B-lymphocyte infection, growth transformation, induction of lytic infection, or virus production. Thus, these experiments also indicate that neither the BHLF1 open reading frame nor the HYG gene insertion critically affects B-lymphocyte infection in vitro.     

Epstein-Barr virus nuclear antigen leader protein induces expression of thymus- and activation-regulated chemokine in B cells

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.     

Use of second-site homologous recombination to demonstrate that Epstein-Barr virus nuclear protein 3B is not important for lymphocyte infection or growth transformation in vitro.

Recombinant Epstein-Barr viruses with a stop codon inserted into the nuclear protein 3B (EBNA 3B) open reading frame were generated by second-site homologous recombination. These mutant viruses infected and growth transformed primary B lymphocytes, resulting in the establishment of lymphoblastoid cell lines (LCLs). Polymerase chain reaction analysis and Southern hybridizations with infected cell DNA demonstrated the presence of the mutant EBNA 3B and the absence of wild-type EBNA 3B. Immunoblot analysis of the LCLs with affinity-purified EBNA 3B antibodies confirmed the absence of EBNA 3B cross-reactive protein. Virus was reactivated from two of these infected LCLs and serially passaged through primary B lymphocytes. The newly infected cells contained only the mutant recombinant virus. No difference was noted between mutant and wild-type recombinants, derived in parallel, in latent (other than EBNA 3B) or lytic cycle-infected cell virus protein expression or in the growth of the latently infected transformed cell lines. These data indicate that the EBNA 3B protein is not critical for primary B-lymphocyte infection, growth transformation, or lytic virus infection in vitro.     

Host cell and EBNA-2 regulation of Epstein-Barr virus latent-cycle promoter activity in B lymphocytes.

The six latent-cycle nuclear antigens (EBNAs) of Epstein-Barr virus (EBV), whose genes share 5' leader exons and two promoters (Cp and Wp), are differentially expressed by cells of the B lineage. To examine the possibility that EBNA gene expression is regulated through selective use of Cp and Wp, we monitored the activity of promoter-chloramphenicol acetyltransferase (CAT) gene constructs transfected into EBV-positive and EBV-negative B lymphocytes and Burkitt's lymphoma cells. Wp was a much stronger promoter than Cp in EBV genome-negative B-cell lines and was used exclusively in primary B cells. When B cells were infected with transforming EBV, Cp became the stronger promoter. This switch was not observed when B cells were infected with an immortalization-deficient virus, P3HR-1, which lacks the EBNA-2 open reading frame and expresses a mutant leader protein (EBNA-LP). Cp function was transactivated when EBV-negative or P3HR-1-infected B cells were cotransfected with Cp and a 12-kb fragment of DNA (BamHI-WWYH) that spanned the P3HR-1 deletion. This activity was mapped to the EBNA-2 gene within WWYH; constructs expressing EBNA-LP did not induce Cp function, and the deletion of 405 bp from the EBNA-2 open reading frame abolished transactivation. This research demonstrates host cell and EBNA-2 regulation of latent-cycle promoter activity in B lymphocytes, a mechanism with implications for persistence of EBV-infected lymphoid cells in vivo.     

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.     

Epstein-Barr virus recombinants with specifically mutated BCRF1 genes.

Epstein-Barr virus (EBV) recombinants with specifically mutated BCRF1 genes were constructed and compared with wild-type BCRF1 recombinants derived in parallel for the ability to initiate and maintain latent infection and growth transformation in primary human B lymphocytes. A stop codon insertion after codon 116 of the 170-codon BCRF1 open reading frame or deletion of the entire gene had no effect on latent infection, B-lymphocyte proliferation into long-term lymphoblastoid cell lines (LCLs), or virus replication. LCLs infected with the stop codon recombinant were indistinguishable from wild-type recombinant-infected LCLs in tumorigenicity in SCID mice. However, mutant BCRF1 recombinant-infected cells differed from wild-type recombinant-infected cells in their inability to block gamma interferon release in cultures of permissively infected LCLs incubated with autologous human peripheral blood mononuclear cells. This is the first functional assay for BCRF1 expression from the EBV genome. BCRF1 probably plays a key role in modulating the specific and nonspecific host responses to EBV infection.     

Epstein-Barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes

Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (DeltaBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the DeltaBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented DeltaBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or DeltaBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a DeltaBNRF1 virus compared to wild-type infection. Likewise, DeltaBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.     

Marker rescue of a transformation-negative Epstein-Barr virus recombinant from an infected Burkitt lymphoma cell line: a method useful for analysis of genes essential for transformation.

A Burkitt lymphoma cell line infected in vitro with a transformation-defective mutant recombinant Epstein-Barr virus (EBV) was used to attempt marker rescue of transformation competence by transfection with cloned wild-type DNA. EBV replication was induced in the transfected cells, and wild-type EBV DNA recombined via flanking homologous sequences adjacent to the deletion, resulting in a virus which transformed primary B lymphocytes in vitro. This strategy should be useful for molecular genetic analysis of the role of part or all of any gene in cell growth transformation.     

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.     

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.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.     

Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.