scholarly journals Integrative profiling of Epstein–Barr virus transcriptome using a multiplatform approach

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Ádám Fülöp ◽  
Gábor Torma ◽  
Norbert Moldován ◽  
Kálmán Szenthe ◽  
Ferenc Bánáti ◽  
...  

Abstract Background Epstein–Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. Methods In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other’s data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. Results This study detected novel genes embedded into longer host genes containing 5′-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. Conclusions An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.

2020 ◽  
Author(s):  
Norbert Moldován ◽  
Kálmán Szenthe ◽  
Ferenc Bánáti ◽  
Ádám Fülöp ◽  
Zsolt Csabai ◽  
...  

Abstract Epstein-Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read- and Pacific Biosciences RS II-based long-read sequencing technologies. In this work, we use the Oxford Nanopore Technologies MinION platform for the characterization of the EBV transcriptomic architecture. Both amplified and non-amplified cDNA sequencings were applied for the generation of transcription reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. This study detected novel short genes (embedded into longer host genes) containing 5’-truncated in-frame open reading frames (ORFs), which might encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel Ori-associated RNA molecules. Additionally, novel mono- and polycistronic, as well as complex transcripts have been uncovered. An intricate meshwork of transcriptional overlaps has also been revealed.


Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 186 ◽  
Author(s):  
Charlotte J. Houldcroft

The nine human herpesviruses are some of the most ubiquitous pathogens worldwide, causing life-long latent infection in a variety of different tissues. Human herpesviruses range from mild childhood infections to known tumour viruses and ‘trolls of transplantation’. Epstein-Barr virus was the first human herpesvirus to have its whole genome sequenced; GenBank now includes thousands of herpesvirus genomes. This review will cover some of the recent advances in our understanding of herpesvirus diversity and disease that have come about as a result of new sequencing technologies, such as target enrichment and long-read sequencing. It will also look at the problem of resolving mixed-genotype infections, whether with short or long-read sequencing methods; and conclude with some thoughts on the future of the field as herpesvirus population genomics becomes a reality.


Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3026-3032 ◽  
Author(s):  
Honglin Chen ◽  
Paul Smith ◽  
Richard F. Ambinder ◽  
S. Diane Hayward

In addition to the Epstein-Barr virus (EBV) EBNA and LMP latency genes, there is a family of alternatively spliced BamHI-A rightward transcripts (BARTs). These latency transcripts are highly expressed in the EBV-associated malignancies nasopharyngeal carcinoma and Burkitt’s lymphoma, and are expressed at lower levels in latently EBV-infected B-cell lines. The contribution of the BARTs to EBV biology or pathogenesis is unknown. Resting B cells have recently been recognized as a reservoir for EBV persistence in the peripheral blood. In these cells, EBV gene expression is tightly restricted and the only viral gene known to be consistently expressed is LMP2A. We used cell sorting and reverse-transcriptase polymerase chain reaction (RT-PCR) to examine whether BARTs are expressed in the restricted form of in vivo latency. Our results demonstrated that RNAs with splicing diagnostic for transcripts containing the BART RPMS1 and BARFO open-reading frames (ORFs) were expressed in CD19+ but not in CD23+ B cells isolated from peripheral blood of healthy individuals. The product of the proximal RPMS1 ORF has not previously been characterized. The RPMS1 ORF was shown to encode a 15-kD protein that localized to the nucleus of transfected cells. Expression of the BARTs in peripheral blood B cells suggests that the proteins encoded by these transcripts are likely to be important for maintenance of in vivo latency.


Virology ◽  
2015 ◽  
Vol 483 ◽  
pp. 44-53 ◽  
Author(s):  
Takahiro Watanabe ◽  
Kenshiro Fuse ◽  
Takahiro Takano ◽  
Yohei Narita ◽  
Fumi Goshima ◽  
...  

2002 ◽  
Vol 76 (1) ◽  
pp. 421-426 ◽  
Author(s):  
Pierre Rivailler ◽  
Hua Jiang ◽  
Young-gyu Cho ◽  
Carol Quink ◽  
Fred Wang

ABSTRACT We sequenced the rhesus lymphocryptovirus (LCV) genome in order to determine its genetic similarity to Epstein-Barr virus (EBV). The rhesus LCV encodes a repertoire identical to that of EBV, with 80 open reading frames, including cellular interleukin-10, bcl-2, and colony-stimulating factor 1 receptor homologues and an equivalent set of viral glycoproteins. The highly conserved rhesus LCV gene repertoire provides a unique animal model for the study of EBV pathogenesis.


2000 ◽  
Vol 74 (7) ◽  
pp. 3082-3092 ◽  
Author(s):  
Paul R. Smith ◽  
Orlando de Jesus ◽  
David Turner ◽  
Martine Hollyoake ◽  
Claudio Elgueta Karstegl ◽  
...  

ABSTRACT CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers. Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open reading frames (ORFs) suggest that, in addition to the much studied BARF0 ORF, there may be important products made from some of the upstream ORFs in the CST RNAs. Potential biological functions are identified for two of these. The product of the RPMS1 ORF is shown to be a nuclear protein that can bind to the CBF1 component of Notch signal transduction. RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2. The protein product of another CST ORF, A73, is shown to be a cytoplasmic protein which can interact with the cell RACK1 protein. Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, the results suggest a possible role for CST products in growth control, perhaps consistent with the abundant transcription of CST RNAs in cancers such as nasopharyngeal carcinoma.


Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3026-3032 ◽  
Author(s):  
Honglin Chen ◽  
Paul Smith ◽  
Richard F. Ambinder ◽  
S. Diane Hayward

Abstract In addition to the Epstein-Barr virus (EBV) EBNA and LMP latency genes, there is a family of alternatively spliced BamHI-A rightward transcripts (BARTs). These latency transcripts are highly expressed in the EBV-associated malignancies nasopharyngeal carcinoma and Burkitt’s lymphoma, and are expressed at lower levels in latently EBV-infected B-cell lines. The contribution of the BARTs to EBV biology or pathogenesis is unknown. Resting B cells have recently been recognized as a reservoir for EBV persistence in the peripheral blood. In these cells, EBV gene expression is tightly restricted and the only viral gene known to be consistently expressed is LMP2A. We used cell sorting and reverse-transcriptase polymerase chain reaction (RT-PCR) to examine whether BARTs are expressed in the restricted form of in vivo latency. Our results demonstrated that RNAs with splicing diagnostic for transcripts containing the BART RPMS1 and BARFO open-reading frames (ORFs) were expressed in CD19+ but not in CD23+ B cells isolated from peripheral blood of healthy individuals. The product of the proximal RPMS1 ORF has not previously been characterized. The RPMS1 ORF was shown to encode a 15-kD protein that localized to the nucleus of transfected cells. Expression of the BARTs in peripheral blood B cells suggests that the proteins encoded by these transcripts are likely to be important for maintenance of in vivo latency.


2018 ◽  
Vol 46 (6) ◽  
pp. 2802-2819 ◽  
Author(s):  
Maja Bencun ◽  
Olaf Klinke ◽  
Agnes Hotz-Wagenblatt ◽  
Severina Klaus ◽  
Ming-Han Tsai ◽  
...  

2003 ◽  
Vol 23 (6) ◽  
pp. 2192-2201 ◽  
Author(s):  
Shao-An Xue ◽  
M. D. Jones ◽  
Qi-Long Lu ◽  
J. M. Middeldorp ◽  
Beverly E. Griffin

ABSTRACT Frameshift mutations provide recognized mechanisms for changing the coding potential of an organism. Here, multiple frameshifts are identified in repetitive sequences within an Epstein-Barr virus unspliced early gene, LF3, which is associated with the viral replicative cycle and also transcriptionally expressed in many virally associated tumors. On the DNA strand encoding LF3, there are three open reading frames, only one of which contains an initiation codon. Most (>95%) of the gene consists of numerous (>20, varying with cell source) GC-rich copies of a 102-bp direct repeat (called IR 4) flanked by small unique sequences. LF3 may express a protein if its initiation and termination codons reside in the same reading frame, but this is not always the case. Frameshifting events, occurring in short runs of pyrimidines (mainly C residues) in the repeats, give rise to mutations which may provide a mechanism for escape of an LF3 function from host surveillance. Sequence studies link these frameshifts to DNA replication errors. Notably, the number of sites in LF3 at which such mutations can occur permits a very large amount of diversity in this gene. Our data also suggest a second degeneracy mechanism within the protein itself, which influences its stability and may reflect a host defense mechanism. LF3 thus provides a potentially important model for studying the quest for supremacy between a virus and its host.


2008 ◽  
Vol 82 (21) ◽  
pp. 10477-10486 ◽  
Author(s):  
Ramakrishna Sompallae ◽  
Stefano Gastaldello ◽  
Sebastian Hildebrand ◽  
Nikolay Zinin ◽  
Gerco Hassink ◽  
...  

ABSTRACT Manipulation of the ubiquitin proteasome system (UPS) is emerging as a common theme in viral pathogenesis. Some viruses have been shown to encode functional homologs of UPS enzymes, suggesting that a systematic identification of these products may provide new insights into virus-host cell interactions. Ubiquitin-specific proteases, collectively known as deubiquitinating enzymes (DUBs), regulate the activity of the UPS by hydrolyzing ubiquitin peptide or isopeptide bonds. The prediction of viral DUBs based on sequence similarity with known enzymes is hampered by the diversity of viral genomes. In this study sequence alignments, pattern searches, and hidden Markov models were developed for the conserved C- and H-boxes of the known DUB families and used to search the open reading frames (ORFs) of Epstein-Barr virus (EBV), a large gammaherpesvirus that has been implicated in the pathogenesis of a broad spectrum of human malignancies of lymphoid and epithelial cell origin. The searches identified a limited number of EBV ORFs that contain putative DUB catalytic domains. DUB activity was confirmed by functional assays and mutation analysis for three high scoring candidates, supporting the usefulness of this bioinformatics approach in predicting distant homologues of cellular enzymes.


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