Bernhard Neuhierl

Glycoprotein gp110 of Epstein–Barr virus determines viral tropism and efficiency of infection

The Epstein–Barr virus (EBV) genome has been detected in lymphomas and in tumors of epithelial or mesenchymal origin such as nasopharyngeal carcinoma or leiomyosarcoma. Thus, there is little doubt that EBV can infect cells of numerous lineages in vivo, in contrast to its in vitro infectious spectrum, which appears restricted predominantly to B lymphocytes. We show here that the EBV BALF4 gene product, the glycoprotein gp110, dramatically enhances the ability of EBV to infect human cells. gp110high viruses were up to 100 times more efficient than their gp110low counterparts in infecting lymphoid or epithelial cells. In addition, gp110high viruses infected the carcinoma cell line HeLa and the T cell lymphoma cell line Molt-4, both previously thought to be refractory to EBV infection. Analysis of several virus isolates showed that the amount of BALF4 present within mature virions markedly differed among these strains. In some strains, gp110 was found expressed during lytic replication not only at the nuclear but also at the cellular membrane. Heterologous expression of gp110 during the virus lytic phase neither altered virus concentration nor affected virus binding to cells. It appears that gp110 plays a crucial role after the virus has adhered to its cellular target. gp110 constitutes an important virulence factor that determines infection of non-B cells by EBV. Therefore, the use of gp110high viruses will help to determine the range of the target cells of EBV beyond B lymphocytes and provide a useful in vitro model to assess the oncogenic potential of EBV in these cells.

Epstein-Barr Virus mRNA Export Factor EB2 Is Essential for Production of Infectious Virus

ABSTRACT 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 (EBVBMLF1-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 EBVBMLF1-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 EBVBMLF1-KO mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.

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.

Epstein-Barr Virus BNRF1 Protein Allows Efficient Transfer from the Endosomal Compartment to the Nucleus of Primary B Lymphocytes

ABSTRACT 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 (ΔBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the ΔBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented ΔBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or ΔBNRF1 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 ΔBNRF1 virus compared to wild-type infection. Likewise, ΔBNRF1 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.

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.

The Epstein-Barr Virus BMRF1 Gene Is Essential for Lytic Virus Replication

ABSTRACT 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 ΔBMRF1 EBV phenotype. ΔBMRF1 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. ΔBMRF1 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.

Primary B-cell infection with a ΔBALF4 Epstein-Barr virus comes to a halt in the endosomal compartment yet still elicits a potent CD4-positive cytotoxic T-cell response

ABSTRACT Epstein-Barr virus (EBV) infection is mediated by several viral envelope glycoproteins. We have assessed gp110s functions during the virus life cycle using a mutant that lacks BALF4 (ΔBALF4). Exposure of various cell lines and primary cell samples of epithelial or lymphoid lineages to the ΔBALF4 mutant failed to establish stable infections. The ΔBALF4 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 ΔBALF4 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 ΔBALF4 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/ΔBALF4 cells. Induction of lymphoblastoid cells generated with transiently complemented ΔBALF4 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 ΔBALF4 viruss 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.