Primary B-cell infection with a deltaBALF4 Epstein-Barr virus comes to a halt in the endosomal compartment yet still elicits a potent CD4-positive cytotoxic T-cell response.

Epstein-Barr virus (EBV) infection is mediated by several viral envelope glycoproteins. We have assessed gp110's functions during the virus life cycle using a mutant that lacks BALF4 (DeltaBALF4). Exposure of various cell lines and primary cell samples of epithelial or lymphoid lineages to the DeltaBALF4 mutant failed to establish stable infections. The DeltaBALF4 virus, however, did not differ from wild-type EBV in its ability to bind and become internalized into primary B cells, in which it elicited a potent T-cell-specific immune reaction against virion constituents. These findings show that DeltaBALF4 viruses can reach the endosome-lysosome compartment and dovetail nicely with the previously identified contribution of gp110 to virus-cell fusion. Other essential steps of the virus life cycle were unaffected in the viral mutant; DNA lytic replication and viral titers were not altered in the absence of gp110, and DeltaBALF4 viruses complemented in trans transformed infected B cells with an efficiency indistinguishable from that observed with wild-type viruses. All of the steps of virus maturation could be observed in lytically induced 293/DeltaBALF4 cells. Induction of lymphoblastoid cells generated with transiently complemented DeltaBALF4 virus led to the production of rare mature virions. We therefore infer that gp110 is not required for virus maturation and egress in 293 cells or in B cells. The DeltaBALF4 virus's phenotypic traits, an inability to infect human cells coupled with potent antigenicity, potentially qualify this mutant as a live vaccine. It will provide a useful tool for the detailed study of EBV-cell interactions in a physiological context.

Epstein-Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection.

Epstein-Barr virus (EBV), a well-characterised B-lymphotropic agent is aetiologically linked to B cell lymphoproliferations, but the spectrum of diseases the virus causes also includes oral hairy leukoplakia, a benign epithelial lesion, as well as carcinomas of the nasopharynx and of the stomach. However, it is still unclear how EBV accesses and transforms primary epithelial cells. Sixteen samples consisting of primary epithelial cells from the sphenoidal sinus or from tonsils were infected with GFP-tagged recombinant B95.8 EBVs produced in the 293 cell line. The rate of infection was assessed by counting GFP-positive cells and cells expressing viral proteins. Primary epithelial cells from all samples were found to be sensitive to EBV infection but there was a marked interindividual variation among the tested samples (2-48% positive cells). This suggests heterogeneity in terms of sensitivity to EBV infection in vivo and therefore possibly to EBV-associated diseases of the epithelium. The virus showed a preferential tropism for differentiated epithelial cells (p63 negative, involucrin positive). In all cases, infected cells expressed EBV lytic proteins but also the LMP1 protein. The viral tropism for differentiated cells and the permissivity of these cells for virus replication reproduced in vitro cardinal features of oral hairy leukoplakia. We have identified a source of EBV that shows unusually strong epitheliotropism for primary epithelial cells that will allow detailed analysis of virus-cell interactions during virus infection, replication and virus-mediated transformation.

The Epstein-Barr virus BMRF1 gene is essential for lytic virus replication.

The Epstein-Barr virus (EBV) BMRF1 protein is a DNA polymerase processivity factor. We have deleted the BMRF1 open reading frame from the EBV genome and assessed the DeltaBMRF1 EBV phenotype. DeltaBMRF1 viruses were replication deficient, but the wild-type phenotype could be restored by BMRF1 trans-complementation. The replication-deficient phenotype included impaired lytic DNA replication and late protein expression. DeltaBMRF1 and wild-type viruses were undistinguishable in terms of their ability to transform primary B cells. Our results provide genetic evidence that BMRF1 is essential for lytic replication of the EBV genome.

Molecular genetics of DNA viruses: recombinant virus technology.

Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.

Overexpression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance and accumulation.

A major goal of phytoremediation is to transform fast-growing plants with genes from plant species that hyperaccumulate toxic trace elements. We overexpressed the gene encoding selenocysteine methyltransferase (SMT) from the selenium (Se) hyperaccumulator Astragalus bisulcatus in Arabidopsis and Indian mustard (Brassica juncea). SMT detoxifies selenocysteine by methylating it to methylselenocysteine, a nonprotein amino acid, thereby diminishing the toxic misincorporation of Se into protein. Our Indian mustard transgenic plants accumulated more Se in the form of methylselenocysteine than the wild type. SMT transgenic seedlings tolerated Se, particularly selenite, significantly better than the wild type, producing 3- to 7-fold greater biomass and 3-fold longer root lengths. Moreover, SMT plants had significantly increased Se accumulation and volatilization. This is the first study, to our knowledge, in which a fast-growing plant was genetically engineered to overexpress a gene from a hyperaccumulator in order to increase phytoremediation potential.

Latent membrane protein 1 is critical for efficient growth transformation of human B cells by epstein-barr virus.

The EBV latent membrane protein 1 (LMP1) is an integral membrane protein that acts like a constitutively activated receptor. LMP1 interacts with members of the tumor necrosis factor receptor-associated factor family, as well as with tumor necrosis factor receptor-associated death domain, resulting in induction of nuclear factor-kappaB, the p38 mitogen-activated protein kinase pathway, and the c-Jun NH(2)-terminal kinase activator protein 1-signaling cascade. The binding of Janus kinase 3 results in activation of signal transducers and activators of transcription. The domain structure of LMP1 has been mapped extensively, but the quantitative contribution of distinct LMP1 domains to the efficiency of B-cell proliferation by EBV has not been determined. On the basis of the maxi-EBV system, which allows us to introduce and study mutations in the context of the complete EBV genome, a panel of 10 EBV mutants with alterations in the LMP1 gene locus was established. The mutant EBVs were tested for their efficiency to induce and maintain proliferation of clonal B-cell lines in vitro. Surprisingly and with reduced frequency, EBV mutants which deleted LMP1's COOH terminus, transmembrane domains, or the entire open reading frame were able to generate proliferating B-cell clones that were dependent on the presence of human fibroblast feeder cells. A B-cell clone carrying the LMP1-null mutant EBV genome was also analyzed for oncogenicity in severe combined immunodeficiency mice. Our results demonstrate that LMP1 is critical but not mandatory for the generation of proliferating B cells in vitro. LMP1 functions greatly contribute to EBV's transformation potential and appear essential for its oncogenicity in severe combined immunodeficiency mice.

Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus.

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.