BHRF1 of Epstein-Barr virus, which is homologous to human proto-oncogene bcl2, is not essential for transformation of B cells or for virus replication in vitro.

The Epstein-Barr virus (EBV) genome contains an open reading frame, BHRF1, that encodes a presumptive membrane protein with sequence similarity to the proto-oncogene bcl2, which is linked to human B-cell follicular lymphoma. Potential roles for BHRF1 in EBV's ability to growth transform human B cells and to replicate in B cells in culture were investigated by generating EBV mutants that lack most of the open reading frame. This was accomplished by recombination of plasmids carrying mutations in BHRF1 with the transformation-defective EBV strain P3HR1. Because BHRF1 resides close to the deletion in P3HR1 that renders this strain transformation defective, B-cell transformation could be used to select for recombination events in the region. B-cell clones were established by recombinants which lacked most of the BHRF1 open reading frame, although most of these initial B-cell transformants also carried nonrecombinant (BHRF1+) P3HR1 genomes, at levels ranging from a fraction of a copy to four copies per cell. Secondary B-cell transformants that lacked BHRF1+ EBV at detectable levels were found to release transforming, BHRF1-deficient EBV at levels that were within the normal range for EBV-immortalized B-cell clones. These studies demonstrate that BHRF1 is nonessential for growth transformation of B cells and for virus replication and release from these cells in culture.     

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.     

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.     

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.     

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

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

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.     

The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation.

These experiments evaluate the role of the Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) in B-lymphocyte growth transformation by using a recombinant EBV molecular genetic approach. Recombinant viruses encoding for a mutant EBNA-LP lacking the carboxy-terminal 45 amino acids were markedly impaired in their ability to transform primary B lymphocytes compared with EBNA-LP wild-type but otherwise isogenic recombinant viruses. This impairment was particularly evident when primary B lymphocytes were infected under conditions of limiting virus dilution. The impairment could be partially corrected by growth of the infected lymphocytes with fibroblast feeder layers or by cocultivation of primary B lymphocytes with relatively highly permissive mutant virus-infected cells. One of the five mutant recombinants recovered by growth of infected cells on fibroblast feeder cultures was a partial revertant which had a normal transforming phenotype. Several lymphoblastoid cell lines infected with the EBNA-LP mutant recombinant viruses had a high percentage of cells with bright cytoplasmic immunoglobulin staining, as is characteristic of cells undergoing plasmacytoid differentiation. Expression of the other EBV latent or lytic proteins and viral replication were not affected by the EBNA-LP mutations. Thus, the EBNA-LP mutant phenotype is not mediated by an effect on expression of another EBV gene. These data are most compatible with the hypothesis that EBNA-LP affects expression of a B-lymphocyte gene which is a mediator of cell growth or differentiation.     

<Emphasis Type="Italic">Herpesvirus pan</Emphasis> encodes a functional homologue of BHRF1, the Epstein-Barr virus v-Bcl-2

Background  

Epstein-Barr virus (EBV) latently infects about 90% of the human population and is associated with benign and malignant diseases of lymphoid and epithelial origin. BHRF1, an early lytic cycle antigen, is an apoptosis suppressing member of the Bcl-2 family. In vitro studies imply that BHRF1 is dispensable for both virus replication and transformation. However, the fact that BHRF1 is highly conserved not only in all EBV isolates studied to date but also in the analogous viruses Herpesvirus papio and Herpesvirus pan that infect baboons and chimpanzees respectively, suggests BHRF1 may play an important role in vivo.     

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term MHC class II presentation of the EBV lytic protein BHRF1 by EBV latently infected b cells following capture of BHRF1 antigen

Although T lymphocytes are considered essential for the control of EBV infection, it remains uncertain how this control occurs. We previously reported unexpected killing of EBV-transformed B-lymphoblastoid cells (LCLs) that did not express BHRF1 by CD4+ T cells specific for BHRF1, an EBV lytic cycle protein. Using LCLs transformed with an EBV mutant, in which the BHRF1 gene was deleted, we showed that killing of latently infected cells through the recognition of a protein produced during the lytic cycle is due to transfer of BHRF1 from lytically infected to latently infected cells, which occurs in culture. Accordingly, LCLs efficiently presented exogenous BHRF1 protein. Furthermore, we present evidence for persistence of captured BHRF1 Ag for several days. Due to this long-term persistence, repeated loading of suboptimal amounts of BHRF1 led to accumulation of BHRF1 Ags in LCLs and, ultimately, to their optimal recognition by BHRF1-specific CD4+ T cells. These results unveil an MHC class II-dependent pathway that could be important for the control of EBV latent infection through recognition of lytic cycle Ags.     

Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.     

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.

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.     

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.     

An Epstein-Barr virus isolated from a lymphoblastoid cell line has a 16-kilobase-pair deletion which includes gp350 and the Epstein-Barr virus nuclear antigen 3A

Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Role of the epstein-barr virus RTA protein in activation of distinct classes of viral lytic cycle genes

Initiation of the Epstein-Barr virus (EBV) lytic cycle is controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta, stimulate their own expression, reciprocally stimulate each other's expression, and activate downstream viral targets. It has been difficult to examine the individual roles of these two transactivators in EBV-infected lymphocytes, as they are expressed simultaneously upon induction of the lytic cycle. Here we show that the Burkitt lymphoma cell line Raji represents an experimental system that allows the study of Rta's role in the lytic cycle of EBV in the absence and presence of ZEBRA. When expressed in Raji cells, exogenous Rta does not activate endogenous BZLF1 expression, yet Rta remains competent to transactivate certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and a late gene, BLRF2, are maximally activated by Rta itself in the absence of detectable ZEBRA. The use of the Z(S186A) mutant form of ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes, such as BMRF1 and BHRF1, that are activated in synergy by Rta and ZEBRA. It has already been documented that of the two activators, only ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA exhibits an inhibitory effect on Rta's capacity to stimulate the late gene, BLRF2. Consequently ZEBRA may function to repress Rta's potential to activate some late genes. Raji cells thus allow delineation of the combinatorial roles of Rta and ZEBRA in control of several distinct classes of lytic cycle genes.     

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.