Helmut Bannert

A Viral microRNA Cluster Strongly Potentiates the Transforming Properties of a Human Herpesvirus

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

The Members of an Epstein-Barr Virus MicroRNA Cluster Cooperate To Transform B Lymphocytes

ABSTRACT Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP). Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBVs oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes in vitro. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miR-BHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miR-BHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells.

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 Alkaline Exonuclease BGLF5 Serves Pleiotropic Functions in Virus Replication

ABSTRACT The Epstein-Barr virus (EBV) alkaline exonuclease BGLF5 has previously been recognized to contribute to immune evasion by downregulating production of HLA molecules during virus replication. We have constructed a BGLF5-null virus mutant to determine BGLF5s functions during EBV viral replication. Quantification of virus production in permissive 293 cells carrying a ΔBGLF5 genome identified a 17- to 21-fold reduction relative to complemented or wild-type controls. Detailed monitoring of ΔBGLF5 replication evidenced an impaired virus nucleocapsid maturation, a reduced primary egress and a 1.4-fold reduction in total viral DNA synthesis. ΔBGLF5 single-unit-length viral genomes were not only less abundant but also migrated faster than expected in gel electrophoresis. We concluded that BGLF5 pertained both to the generation and to the processing of viral linear genomes. ΔBGLF5 phenotypic traits were reminiscent of those previously identified in a mutant devoid of UL12, BGLF5s homolog in herpes simplex virus type 1, and indeed UL12 was found to partially complement the ΔBGLF5 phenotype. However, BGLF5-specific functions could also be identified; the nuclear membrane of replicating cells displayed images of reduplication and complex folding that could be completely corrected by BGLF5 but not UL12. Similar nuclear abnormalities were previously observed in cells transfected with BFLF2 and BFRF1, two viral proteins crucial for EBV nuclear egress. Interestingly, ΔBGLF5 cells produced more BFLF2 than wild-type or complemented counterparts. The present study provides an overview of BGLF5s functions that will guide future molecular studies. We anticipate that the 293/ΔBGLF5 cell line will be instrumental in such developments.

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.

The Epstein-Barr Virus Protein Kinase BGLF4 and the Exonuclease BGLF5 Have Opposite Effects on the Regulation of Viral Protein Production

ABSTRACT The Epstein-Barr virus BGLF4 and BGLF5 genes encode a protein kinase and an alkaline exonuclease, respectively. Both proteins were previously found to regulate multiple steps of virus replication, including lytic DNA replication and primary egress. However, while inactivation of BGLF4 led to the downregulation of several viral proteins, the absence of BGLF5 had the opposite effect. Using recombinant viruses that lack both viral enzymes, we confirm and extend these initial observations, e.g., by showing that both BGLF4 and BGLF5 are required for proper phosphorylation of the DNA polymerase processivity factor BMRF1. We further found that neither BGLF4 nor BGLF5 is required for baseline viral protein production. Complementation with BGLF5 downregulated mRNA levels and translation of numerous viral genes, though to various degrees, whereas BGLF4 had the opposite effect. BGLF4 and BGLF5 influences on viral expression were most pronounced for BFRF1 and BFLF2, two proteins essential for nuclear egress. For most viral genes studied, cotransfection of BGLF4 and BGLF5 had only a marginal influence on their expression patterns, showing that BGLF4 antagonizes BGLF5-mediated viral gene shutoff. To be able to exert its functions on viral gene expression, BGLF4 must be able to escape BGLF5s shutoff activities. Indeed, we found that BGLF5 stimulated the BGLF4 genes transcription through an as yet uncharacterized molecular mechanism. The BGLF4/BGLF5 enzyme pair builds a regulatory loop that allows fine-tuning of virus protein production, which is required for efficient viral replication.

The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells

The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects the majority of the human population. All EBV strains transform B lymphocytes, but some strains, such as M81, also induce spontaneous virus replication. EBV encodes 22 microRNAs (miRNAs) that form a cluster within the BART region of the virus and have been previously been found to stimulate tumor cell growth. Here we describe their functions in B cells infected by M81. We found that the BART miRNAs are downregulated in replicating cells, and that exposure of B cells in vitro or in vivo in humanized mice to a BART miRNA knockout virus resulted in an increased proportion of spontaneously replicating cells, relative to wild type virus. The BART miRNAs subcluster 1, and to a lesser extent subcluster 2, prevented expression of BZLF1, the key protein for initiation of lytic replication. Thus, multiple BART miRNAs cooperate to repress lytic replication. The BART miRNAs also downregulated pro- and anti-apoptotic mediators such as caspase 3 and LMP1, and their deletion did not sensitize B-cells to apoptosis. To the contrary, the majority of humanized mice infected with the BART miRNA knockout mutant developed tumors more rapidly, probably due to enhanced LMP1 expression, although deletion of the BART miRNAs did not modify the virus transforming abilities in vitro. This ability to slow cell growth could be confirmed in non-humanized immunocompromized mice. Injection of resting B cells exposed to a virus that lacks the BART miRNAs resulted in accelerated tumor growth, relative to wild type controls. Therefore, we found that the M81 BART miRNAs do not enhance B-cell tumorigenesis but rather repress it. The repressive effects of the BART miRNAs on potentially pathogenic viral functions in infected B cells are likely to facilitate long-term persistence of the virus in the infected host.