Platinum-carbide interactions: core-shells for catalytic use.

A selection of carbides (TiC, NbC, TaC, WC and SiC) have been studied using density functional theory. Their calculated adsorption characteristics towards Pt overlayers are presented with a view to the use of the carbides as core-shell components. WC hcp and SiC are observed to support Pt overlayers on all surfaces making them promising candidates for full Pt encapsulation. Pt adsorption on fcc (111) carbide surfaces is observed to take place with the transition metal surface resonances (TMSRs) playing a key role whilst fcc (100) are universally unfavourable towards Pt adsorption. The effect of the carbide supports on oxygen binding on the Pt overlayer is also considered and discussed in relation to the oxygen reduction reaction (ORR). The oxygen adsorption study revealed several Pt-WC surfaces which exhibit reduced oxygen adsorption energies, suggesting they should promote or maintain ORR activity with respect to nanoparticulate Pt catalysts.

The Yin and Yang of inflammation.

Inflammation is an essential protective part of the body's response to infection, yet many diseases are the product of inflammation. For example, inflammation can lead to autoimmune disease and tissue damage, and is a key element in chronic health conditions such as heart disease, diabetes, rheumatoid arthritis, and also drives changes associated with aging. Animal models of infectious and chronic disease are important tools with which to dissect the pathways whereby inflammatory responses are initiated and controlled. Animal models therefore provide a prism through which the role of inflammation in health and disease can be viewed, and are important means by which to dissect mechanisms and identify potential therapies to be tested in the clinic. A meeting, "The Yin and Yang of Inflammation" was organized by Trudeau Institute and was held between April 4-6, 2014. The main goal was to bring together experts from biotechnology and academic organizations to examine and describe critical pathways in inflammation and place these pathways within the context of human disease. A group of ~80 scientists met for three days of intense formal and informal exchanges. A key focus was to stimulate interactions between basic research and industry.

Fine mapping of the nematode resistance gene Mi-3 in Solanum peruvianum and construction of a S. lycopersicum DNA contig spanning the locus.

Currently, the only genetic resistance against root-knot nematodes in the cultivated tomato Solanum lycopersicum (Lycopersicon esculentum) is due to the gene Mi-1. Another resistance gene, Mi-3, identified in the related wild species Solanum peruvianum (Lycopersicon peruvianum) confers resistance to nematodes that are virulent on tomato lines that carry Mi-1, and is effective at temperatures at which Mi-1 is not effective (above 30 degrees C). Two S. peruvianum populations segregating for Mi-3 were used to develop a high-resolution map of the Mi-3 region of chromosome 12. S. lycopersicum BACs carrying flanking markers were identified and used to construct a contig spanning the Mi-3 region. Markers generated from BAC-end sequences were mapped in S. peruvianum plants in which recombination events had occurred near Mi-3. Comparison of the S. peruvianum genetic map with the physical map of S. lycopersicum indicated that marker order is conserved between S. lycopersicum and S. peruvianum. The 600 kb contig between Mi-3-flanking markers TG180 and NR18 corresponds to a genetic distance of about 7.2 cM in S. peruvianum. We have identified a marker that completely cosegregates with Mi-3, as well as flanking markers within 0.25 cM of the gene. These markers can be used to introduce Mi-3 into cultivated tomato, either by conventional breeding or cloning strategies.

Replication from oriP of Epstein-Barr virus requires exact spacing of two bound dimers of EBNA1 which bend DNA.

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on oriP are replicated once per cell cycle by cellular factors. The replicator of oriP is an approximately 120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from oriP requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.

Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein-Barr virus.

Epstein-Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP-dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co-immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.

Human DNA replication initiation factors, ORC and MCM, associate with oriP of Epstein-Barr virus.

The 165-kb chromosome of Epstein-Barr virus (EBV) is replicated by cellular enzymes only once per cell cycle in human cells that are latently infected. Here, we report that the human origin recognition complex, ORC, can be detected in association with an EBV replication origin, oriP, in cells by using antibodies against three different subunits of human ORC to precipitate crosslinked chromatin. Mcm2, a subunit of the MCM replication licensing complex, was found to associate with oriP during G(1) and to dissociate from it during S phase. The detection of ORC and Mcm2 at oriP was shown to require the presence of the 120-bp replicator of oriP. Licensing and initiation of replication at oriP of EBV thus seem to be mediated by ORC. This is an example of a virus apparently using ORC and associated factors for the propagation of its genome.

Initiation of DNA replication within oriP is dispensable for stable replication of the latent Epstein-Barr virus chromosome after infection of established cell lines.

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called oriP, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of oriP, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of oriP, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the oriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of oriP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of oriP. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.

The minimal replicator of Epstein-Barr virus oriP.

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells. oriP contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of oriP, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from oriP per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying oriP required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each "half" of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the "left half" and once in the "right half." These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of oriP is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites.

Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus.

Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1, 800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

CD21-Dependent infection of an epithelial cell line, 293, by Epstein-Barr virus.

Epstein-Barr virus (EBV) is invariably present in undifferentiated nasopharyngeal carcinomas, is found sporadically in other carcinomas, and replicates in the differentiated layer of the tongue epithelium in lesions of oral hairy leukoplakia. However, it is not clear how frequently or by what mechanism EBV infects epithelial cells normally. Here, we report that a human epithelial cell line, 293, can be stably infected by EBV that has been genetically marked with a selectable gene. We show that 293 cells express a relatively low level of CD21, that binding of fluorescein-labeled EBV to 293 cells can be detected, and that both the binding of virus to cells and infection can be blocked with antibodies specific for CD21. Two proteins known to form complexes with CD21 on the surface of lymphoid cells, CD35 and CD19, could not be detected at the surface of 293 cells. All infected clones of 293 cells exhibited tight latency with a pattern of gene expression similar to that of type II latency, but productive EBV replication and release of infectious virus could be induced inefficiently by forced expression of the lytic transactivators, R and Z. Low levels of mRNA specific for the transforming membrane protein of EBV, LMP-1, as well as for LMP-2, were detected; however, LMP-1 protein was either undetectable or near the limit of detection at less than 5% of the level typical of EBV-transformed B cells. A slight increase in expression of the receptor for epidermal growth factor, which can be induced in epithelial cells by LMP-1, was detected at the cell surface with two EBV-infected 293 cell clones. These results show that low levels of surface CD21 can support infection of an epithelial cell line by EBV. The results also raise the possibility that in a normal infection of epithelial cells by EBV, the LMP-1 protein is not expressed at levels that are high enough to be oncogenic and that there might be differences in the cells of EBV-associated epithelial cancers that have arisen to allow for elevated expression of LMP-1.

Characterization of accessory molecules in murine lung dendritic cell function: roles for CD80, CD86, CD54, and CD40L.

Lung dendritic cells (DCs) from mice were enriched to 92-99% purity using a multistep enrichment protocol which included fluorescence-activated cell sorting. DCs were analyzed for expression of cell surface molecules, function in a mixed leukocyte reaction (MLR), and dependence on accessory molecules in stimulating an MLR. DCs possessed potent accessory properties in vitro, while interstitial macrophages (IM), which are Ia-negative, displayed little MLR-stimulating function of their own. However, IM were capable of enhancing DC-initiated T cell proliferation via a cell contact mechanism. These results indicated that murine lung DCs functioned as stimulators of primary T cell responses, and that cells in the local environment influenced their function. Lung DCs expressed surface molecules typical of DCs from other sites including CD11a, CD54, CD80, and CD86. As is true for DCs in other sites, costimulatory molecules including CD80, CD86, CD40L, CD2, CD54, and CD11a played important roles in lung DC-initiated T cell proliferation. Interestingly, anti-CD86 monoclonal antibody (mAb) had little inhibitory effect on the MLR unless it was added in combination with anti-CD80 mAb. These studies suggest that CD80 on lung DCs can provide a costimulatory signal to allogeneic T cells in the absence of CD86 signaling, but that CD86 functions poorly except when CD80 is also engaged.

The failed Patient Self-Determination Act and policy alternatives for the right to die.

The empirical evidence regarding the implementation and impact of the federal Patient Self-Determination Act is examined in this article. The Act was designed to increase the use of advance medical directives in light of the U.S. Supreme Court's Cruzan decision. Research shows that the law has had little effect and that the use of advance directives has scant relation to medical treatment and care. Various policy alternatives for the right to die are also examined. The authors conclude with an analysis of the likely impact of medical costs, fruitless treatment, and rationed health care on limiting life-prolonging treatment.

An imperfect correlation between DNA replication activity of Epstein-Barr virus nuclear antigen 1 (EBNA1) and binding to the nuclear import receptor, Rch1/importin alpha.

Epstein-Barr virus (EBV) replicates as a stable multicopy episome in latently infected mammalian cells. Latent cycle DNA replication requires only two viral elements, the cis-acting origin of plasmid replication (oriP) and the trans-acting origin binding protein (EBNA1). EBNA1 binds multiple recognition sites in oriP, but has not other enzymatic activities associated with replication functions. To identify human cellular proteins that mediate EBNA1 function, we designed a one-hybrid assay in yeast to select for proteins that bind to EBNA1 when bound to criP in vivo. A human cDNA encoding the Rch1/hSRP1 alpha/ importin alpha protein was isolated and shown to bind to full-length EBNA1, but not to an amino terminal deletion mutant of EBNA1 when bound to oriP in yeast. The interaction of EBNA1 with Rch1 was confirmed biochemically by coimmunoprecipitation from nuclear extracts and by direct binding of recombinant proteins in vitro. Internal deletion mutations in EBNA1 which compromised DNA replication activity were similarly reduced for binding to Rch1. Mutations with no effect on DNA replication activity were similarly unaffected for Rch1 binding. Rch1/importin alpha has been shown to bind to the nuclear localization sequence (NLS) of several proteins and stimulate nuclear import. A substitution mutation in the EBNA1 nuclear localization sequence reduced Rch1 binding, but had no effect on DNA replication function, indicating that Rch1 binding affinity does not correspond precisely with replication activity. Nevertheless, the identification of a stable interaction between Rch1 and EBNA1 at the origin of viral DNA replication raises the intriguing possibility that Rch1 contributes to the nuclear functions of EBNA1.

Comparison of the EBNA1 proteins of Epstein-Barr virus and herpesvirus papio in sequence and function.

The EBNA1 protein of Epstein-Barr virus (EBV) supports replication and maintenance of the circularized viral chromosome in cells that are latently infected. We have isolated, sequenced, and functionally characterized the EBNA1 gene of herpesvirus papio (HVP), an EBV-like virus that infects baboons. The amino acid sequences of EBNA1 of HVP and EBV are 56% identical, if the difference in the length of the glycine and alanine containing repetitive region, which is much shorter for HVP EBNA1, is omitted for the calculation. The key structural features of the DNA-binding/dimerization domain (the carboxyl-terminal domain) appear to have been conserved, as have amino acids in the two regions thought to be most critical for DNA binding. Most of the salient features of the amino-terminal two-thirds of EBNA1 (the amino-terminal domain), including a dearth of sequences predictive of alpha-helical or beta-sheet structures, are shared by the two sequences, although numerous gaps in this region were needed for alignment of the sequences. The amino-terminal fifty amino acids of EBNA1 of both EBV and HVP weakly resemble the amino terminus of rat ribosomal protein S2. Plasmids carrying oriP of either virus replicated stably in mammalian cells and supported efficient outgrowth of colonies under selection when supported by EBNA1 from either virus, although with each oriP there was a noticeable preference for EBNA1 to be from the same virus. HVP EBNA1 was less effective than EBV EBNA1 at activating the enhancer function of EBV oriP and under certain conditions was less effective than EBV EBNA1 at supporting maintenance of plasmids carrying EBV oriP. Results obtained with hybrid EBNA1 molecules indicated that differences in the amino-terminal and carboxyl-terminal domains, respectively, are primarily responsible for the differences in transcriptional activation and plasmid maintenance, respectively. The results showed that changes within EBNA1 can differentially alter its transcriptional and replicational activities.

Mutants of Epstein-Barr virus with a selective marker disrupting the TP gene transform B cells and replicate normally in culture.

We have isolated mutants of Epstein-Barr virus (EBV) which carry a dominant selectable marker inserted into the third exon of the gene encoding two membrane proteins, TP1 and TP2 (or LMP2A and LMP2B), which are expressed in latently infected, growth-transformed B cells. One of the mutants also acquired a 260-bp deletion beginning in the first intron a few base pairs from the terminal repeats and removing most of the second TP exon, including the initial coding sequences of TP2. These EBV mutants transform human B cells in culture, and the transformed B-cell clones carrying them release EBV at approximately normal frequencies.

The role of T lymphocytes in pulmonary microbial defense mechanisms.

Understanding how lung immunity develops against pulmonary pathogens should lead to more rational approaches in vaccine design and to the use of recombinant cytokines in lung disease. T lymphocytes are central to the development of effective immune responses; therefore, understanding how lung immunity develops will require a study of how and where T cells respond to respiratory antigens. Our laboratory has helped define the phenotype and function of lung dendritic cells, which likely play an essential role in stimulating naive T cells to respond to antigens. We found that both interstitial and alveolar macrophages can regulate the function of these cells, the former to enhance activity, the latter to suppress. In addition, we developed a murine pulmonary infection model using the fungus, Cryptococcus neoformans, in which T-cell-mediated immunity is essential for effective host clearance of the organism. The role of T cells in this model is to recruit and activate effector cells to resolve the lung infection; both CD4 and CD8 T-cell subsets are required for optimal effector cell recruitment. These studies are summarized as examples of current approaches to understanding pulmonary immunity.

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.

A role for TGF-beta in the suppression by murine bronchoalveolar cells of lung dendritic cell initiated immune responses.

Effective pulmonary immune responses likely require both local antigen presenting cells (APC) and regulatory suppressor cells. Bronchoalveolar cells (BAC), which consist primarily of alveolar macrophages (AM), are poor APC in most species and are often suppressive. However, dendritic cell (DC)-enriched populations from both lung interstitium and BAC have potent APC activity as measured by their capacity to stimulate both alloantigen and antigen-induced lymphoproliferative T cell responses. To determine if BAC could down-regulate pulmonary immune responses, BAC were mixed with DC-enriched loosely adherent lung interstitial cells (LAd) in a mixed leukocyte reaction (MLR). With high numbers of BAC, MLRs were consistently suppressed and suppression was partially reversed by the addition of indomethacin and catalase. Supernatants from BAC cultured with either syngeneic or allogeneic T lymphocytes in the presence of indomethacin and catalase markedly suppressed an MLR, while supernatants from BAC cultured alone were inconsistently suppressive. Antibodies to TGF-beta completely reversed the BAC-T cell supernatant-induced suppression of the MLR. However, TGF-beta antibody only partially reversed BAC-induced suppression when BAC were added directly to MLR cultures that contained indomethacin and catalase, suggesting that, in addition to TGF-beta, prostaglandins, and H2O2, BAC in culture with LAd and allogeneic T cells also produced short-lived suppressive factors and/or mediated suppression by direct cell contact. Thus, resident BAC likely utilize multiple mechanisms including TGF-beta secretion to suppress intra-alveolar immune responses initiated by lung DC.

Targeted gene disruption in Epstein-Barr virus.

We report the development of a method that should allow the insertion of a selective marker into any region of the Epstein-Barr virus (EBV) genome of strain B95-8 through homologous recombination with plasmids. In this method, EBV recombinants are isolated as G418-resistant, immortalized B-cell clones or as G418-resistant, latently infected subclones of Burkitt lymphoma cell lines. The presence of the productive replication origin of EBV, oriLyt, on the plasmid was found to increase the number of observed recombinant viruses by approximately 100-fold; this stimulation was observed when oriLyt was separated from the sites of recombination by several kilobases of nonhomologous DNA. Long segments of EBV DNA flanking the marker on the plasmid and/or a large plasmid size were inferred to be important for obtaining a high proportion of recombinant genomes that had recombined on both sides of the selective marker; otherwise, the recombinants that predominated had acquired the entire plasmid by recombining only on one side of the inserted marker. Therefore, to facilitate targeted insertion of genetic markers into the EBV genome, a cosmid vector carrying oriLyt was constructed and tested by using it to generate EBV mutants with the BALF2 open-reading frame disrupted.

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.