Epstein-Barr virus genetics: talking about the BAC generation

Genetic mutant organisms pervade all areas of Biology. Early on, herpesviruses (HV) were found to be amenable to genetic analysis using homologous recombination techniques in eukaryotic cells. More recently, HV genomes cloned onto a bacterial artificial chromosome (BAC) have become available. HV BACs can be easily modified in E.coli and reintroduced in eukaryotic cells to produce infectious viruses. Mutants derived from HV BACs have been used both to understand the functions of all types of genetic elements present on the virus genome, but also to generate mutants with potentially medically relevant properties such as preventative vaccines. Here we retrace the development of the BAC technology applied to the Epstein-Barr virus (EBV) and review the strategies available for the construction of mutants. We expand on the appropriate controls required for proper use of the EBV BACs, and on the technical hurdles researchers face in working with these recombinants. We then discuss how further technological developments might successfully overcome these difficulties. Finally, we catalog the EBV BAC mutants that are currently available and illustrate their contributions to the field using a few representative examples

Progress in EBV Vaccines

The Epstein-Barr virus (EBV) is a ubiquitous pathogen that imparts a significant burden of disease on the human population. EBV is the primary cause of infectious mononucleosis and is etiologically linked to the development of numerous malignancies. In recent years, evidence has also been amassed that strongly implicate EBV in the development of several autoimmune diseases, including multiple sclerosis. Prophylactic and therapeutic vaccination has been touted as a possible means of preventing EBV infection and controlling EBV-associated diseases. However, despite several decades of research, no licensed EBV vaccine is available. The majority of EBV vaccination studies over the last two decades have focused on the major envelope protein gp350, culminating in a phase II clinical trial that showed soluble gp350 reduced the incidence of IM, although it was unable to protect against EBV infection. Recently, novel vaccine candidates with increased structural complexity and antigenic content have been developed. The ability of next generation vaccines to safeguard against B-cell and epithelial cell infection, as well as to target infected cells during all phases of infection, is likely to decrease the negative impact of EBV infection on the human population

Epstein-Barr Virus Infection of Naïve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD− CD27+ switched memory (SM) B cells, not in IgD+ CD27− naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes

EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus

Expression and Processing of a Small Nucleolar RNA from the Epstein-Barr Virus Genome

Small nucleolar RNAs (snoRNAs) are localized within the nucleolus, a sub-nuclear compartment, in which they guide ribosomal or spliceosomal RNA modifications, respectively. Up until now, snoRNAs have only been identified in eukaryal and archaeal genomes, but are notably absent in bacteria. By screening B lymphocytes for expression of non-coding RNAs (ncRNAs) induced by the Epstein-Barr virus (EBV), we here report, for the first time, the identification of a snoRNA gene within a viral genome, designated as v-snoRNA1. This genetic element displays all hallmark sequence motifs of a canonical C/D box snoRNA, namely C/C′- as well as D/D′-boxes. The nucleolar localization of v-snoRNA1 was verified by in situ hybridisation of EBV-infected cells. We also confirmed binding of the three canonical snoRNA proteins, fibrillarin, Nop56 and Nop58, to v-snoRNA1. The C-box motif of v-snoRNA1 was shown to be crucial for the stability of the viral snoRNA; its selective deletion in the viral genome led to a complete down-regulation of v-snoRNA1 expression levels within EBV-infected B cells. We further provide evidence that v-snoRNA1 might serve as a miRNA-like precursor, which is processed into 24 nt sized RNA species, designated as v-snoRNA124pp. A potential target site of v-snoRNA124pp was identified within the 3′-UTR of BALF5 mRNA which encodes the viral DNA polymerase. V-snoRNA1 was found to be expressed in all investigated EBV-positive cell lines, including lymphoblastoid cell lines (LCL). Interestingly, induction of the lytic cycle markedly up-regulated expression levels of v-snoRNA1 up to 30-fold. By a computational approach, we identified a v-snoRNA1 homolog in the rhesus lymphocryptovirus genome. This evolutionary conservation suggests an important role of v-snoRNA1 during γ-herpesvirus infection

The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3′UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3′UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies

Epstein-Barr virus infection and human malignancies

The Epstein-Barr virus (EBV) is a herpes virus which establishes a life-long persistent infection in over 90% of the human adult population world-wide. Based on its association with a variety of lymphoid and epithelial malignancies, EBV has been classified as a group 1 carcinogen by the International Agency for Research on Cancer. In this article we discuss the evidence supporting an aetiological role for EBV in the pathogenesis of human tumours. The biology of EBV infection will be described with special emphasis on viral transforming gene products. A brief survey of EBV-associated tumours is followed by a discussion of specific problems. Evidence is presented which suggests that failures of the EBV-specific immunity may play a role in the pathogenesis of EBV-associated tumours also in patients without clinically manifest immunodeficiencies. Finally, the timing of EBV infection in the pathogenesis of virus-associated malignancies is discussed. There is good evidence that EBV infection precedes expansion of the malignant cell populations in some virus-associated tumours. However, this is clearly not always the case and for some of these tumours there are indications that clonal genetic alterations may occur prior to EBV infection. Thus, whilst there is good evidence to suggest that EBV is a human carcinogen, its precise role(s) in the development of virus-associated human tumours requires clarification

Epstein-Barr virus infectious particles initiate B cell transformation and modulate cytokine response

ABSTRACT The Epstein-Barr virus (EBV) efficiently transforms primary B cells. Here, we show that this process starts immediately after cellular exposure to infectious viral particles. Virus binding to B cells led to the activation of intracytoplasmic tyrosine kinases and STAT3. Tegument proteins within the virion in turn activated the p38-MK2 pathway upon cell entry, independently of the viral DNA. Engagement of STAT3 and p38/MK2, two pro-inflammatory pathways, was essential for expression of the key EBV transforming gene EBNA2 but also facilitated IL-6 and TNFα release. However, these pathways simultaneously activated ZFP36L1, a stress response protein that targets transcripts with an AU-rich 3′UTR, to reduce IL-6 and TNFα transcription in infected cells. Expression of viral latent proteins after infection amplified the viral effects on p38 and MK2, but also on ZFP36L1, altogether resulting in a transitory and limited increase in IL-6 and TNFα transcription and release. Thus, EBV virions are not merely vehicles that allow injection of the viral DNA into the nucleus but manipulate cellular pathways to initiate transformation while limiting cytokine release. IMPORTANCE The Epstein-Barr virus efficiently infects and transforms B lymphocytes. During this process, infectious viral particles transport the viral genome to the nucleus of target cells. We show here that these complex viral structures serve additional crucial roles by activating transcription of the transforming genes encoded by the virus. We show that components of the infectious particle sequentially activate proinflammatory B lymphocyte signaling pathways that, in turn, activate viral gene expression but also cause cytokine release. However, virus infection activates expression of ZFP36L1, an RNA-binding stress protein that limits the length and the intensity of the cytokine response. Thus, the infectious particles can activate viral gene expression and initiate cellular transformation at the price of a limited immune response