Translational profiling of B cells infected with the Epstein-Barr virus reveals 5' leader ribosome recruitment through upstream open reading frames.

The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs. A similar process was visible for a subset of lytic genes including the key transactivators BZLF1 and BRLF1 in cells infected with weakly replicating EBV strains. This suggests that ribosome trapping, particularly in the leader region, represents a new checkpoint for the repression of lytic replication. We could identify 25 upstream open reading frames (uORFs) located upstream of coding transcripts that displayed 5' leader ribosome trapping, six of which were located in the leader region shared by many latent transcripts. These uORFs repressed viral translation and are likely to play an important role in the regulation of EBV translation.

Epstein-Barr Virus: From the Detection of Sequence Polymorphisms to the Recognition of Viral Types.

The Epstein-Barr virus is etiologically linked with the development of benign and malignant diseases, characterized by their diversity and a heterogeneous geographic distribution across the world. The virus possesses a 170-kb-large genome that encodes for multiple proteins and non-coding RNAs. Early on there have been numerous attempts to link particular diseases with particular EBV strains, or at least with viral genetic polymorphisms. This has given rise to a wealth of information whose value has been difficult to evaluate for at least four reasons. First, most studies have looked only at one particular gene and missed the global picture. Second, they usually have not studied sufficient numbers of diseased and control cases to reach robust statistical significance. Third, the functional significance of most polymorphisms has remained unclear, although there are exceptions such as the 30-bp deletion in LMP1. Fourth, different biological properties of the virus do not necessarily equate with a different pathogenicity. This was best illustrated by the type 1 and type 2 viruses that markedly differ in terms of their transformation abilities, yet do not seem to cause different diseases. Reciprocally, environmental and genetic factors in the host are likely to influence the outcome of infections with the same virus type. However, with recent developments in recombinant virus technology and in the availability of high throughput sequencing, the tide is now turning. The availability of 23 complete or nearly complete genomes has led to the recognition of viral subtypes, some of which possess nearly identical genotypes. Furthermore, there is growing evidence that some genetic polymorphisms among EBV strains markedly influence the biological and clinical behavior of the virus. Some virus strains are endowed with biological properties that explain crucial clinical features of patients with EBV-associated diseases. Although we now have a better overview of the genetic diversity within EBV genomes, it has also become clear that defining phenotypic traits evinced by cells infected by different viruses usually result from the combination of multiple polymorphisms that will be difficult to identify in their entirety. However, the steadily increasing number of sequenced EBV genomes and cloned EBV BACS from diseased and healthy patients will facilitate the identification of the key polymorphisms that condition the biological and clinical behavior of the viruses. This will allow the development of preventative and therapeutic approaches against highly pathogenic viral strains.

KIT Mutation and Loss of 14q May Be Sufficient for the Development of Clinically Symptomatic Very Low-Risk GIST.

The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.

Genetics of Epstein-Barr virus microRNAs.

The Epstein-Barr virus encodes at least 44 microRNAs that are grouped in two clusters located around the BHRF1 gene and within the BART transcript. The expression pattern of these microRNAs both depends on the lineage of the infected cells and on the type of viral latency. Whilst BART microRNAs are expressed in all EBV-infected tumors, the BHRF1 locus is nearly exclusively expressed in cells that display a type III latency. However, the BART microRNA expression level is several orders of magnitude higher in epithelial cells than in B cells. Genetic studies have demonstrated that the BHRF1 microRNA cluster enhances the initial phases of primary B cell transformation through inhibition of apoptosis. A similar role has been ascribed to the BART microRNAs although their contribution to this process seems more limited. These microRNAs also enhance the survival of B cell lymphoma cells. Using various strategies including high throughput assays, several groups have identified mRNAs targeted by the EBV microRNAs. Here we compare the results of the published high throughput screens and review the viral and cellular genes thought to represent high confidence targets for the EBV microRNAs. Although genetic studies allow unequivocal evaluation of the functions served by the microRNAs, only a few key targets have been identified so far.

Spontaneous lytic replication and epitheliotropism define an Epstein-Barr virus strain found in carcinomas.

The Epstein-Barr virus (EBV) is found in a variety of tumors whose incidence greatly varies around the world. A poorly explored hypothesis is that particular EBV strains account for this phenomenon. We report that M81, a virus isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), shows remarkable similarity to other NPC viruses but is divergent from all other known strains. M81 exhibited a reversed tropism relative to common strains with a reduced ability to infect B cells and a high propensity to infect epithelial cells, which is in agreement with its isolation from carcinomas. M81 spontaneously replicated in B cells in vitro and in vivo at unusually high levels, in line with the enhanced viral replication observed in NPC patients. Spontaneous replication and epitheliotropism could be partly ascribed to polymorphisms within viral proteins. We suggest considering M81 and its closely related isolates as an EBV subtype with enhanced pathogenic potential.