scholarly journals High efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells

2018 ◽  
Author(s):  
Andrew D. Johnston ◽  
Claudia A. Simões-Pires ◽  
Masako Suzuki ◽  
John M. Greally
Keyword(s):  
High Efficiency ◽  
Single Cells ◽  
Amplicon Sequencing ◽  
Barr Virus ◽  
Functional Variant ◽  
Pcr Products ◽  
Flow Cytometric Sorting ◽  
Epstein Barr ◽  
Long Range Pcr ◽  
Genomic Editing

ABSTRACTWhile lymphoblastoid cell lines (LCLs) represent a valuable resource for population genetic studies, they are usually regarded as difficult for CRISPR-mediated genomic editing. It would be valuable to be able to take the results of their functional variant studies and test them in the same LCLs. We describe a protocol using a single-stranded donor oligonucleotide (ssODN) strategy for ‘scarless’ editing in LCLs. The protocol involves optimized transfection, flow cytometric sorting of transfected cells to single cells in multi-well plates and growth in conditioned, serum-rich medium, followed by characterization of the clones. Amplicon sequencing reveals the relative proportions of alleles with different editing events, with sequencing of DNA from clones showing the frequencies of events in individual cells. We find 12/60 (20%) of clones selected in this manner to have the desired ssODN-mediated recombination event. Long-range PCR of DNA at the edited locus and of RT-PCR products for the gene traversing the edited locus reveals 3/6 characterized clones (50%) to have large structural mutations of the region that are missed by sequencing just the edited site. The protocol does not require the use of lentiviruses or stable transfection, and makes LCLs a realistic cell type for consideration for CRISPR-mediated genomic targeting.

scholarly journals Points of Recombination in Epstein-Barr Virus (EBV) Strain P3HR-1-Derived Heterogeneous DNA as Indexes to EBV DNA Recombinogenic Events In Vivo

2008 ◽  
Vol 82 (23) ◽  
pp. 11516-11525 ◽  
Author(s):  
Kazufumi Ikuta ◽  
Shamala K. Srinivas ◽  
Tim Schacker ◽  
Jun-ichi Miyagi ◽  
Rona S. Scott ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Lymphoid Cells ◽  
Defective Interfering Particles ◽  
Barr Virus ◽  
Ebv Reactivation ◽  
Pcr Products ◽  
Ebv Dna ◽  
Epstein Barr

ABSTRACT Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation.

scholarly journals Upregulation of STAT3 Marks Burkitt Lymphoma Cells Refractory to Epstein-Barr Virus Lytic Cycle Induction by HDAC Inhibitors

2009 ◽  
Vol 84 (2) ◽  
pp. 993-1004 ◽  
Author(s):  
Derek Daigle ◽  
Cynthia Megyola ◽  
Ayman El-Guindy ◽  
Lyn Gradoville ◽  
David Tuck ◽  
...  
Keyword(s):  
Burkitt Lymphoma ◽  
Epstein Barr Virus ◽  
De Novo ◽  
Fundamental Problem ◽  
Single Cells ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Refractory State ◽  
Epstein Barr

ABSTRACT A fundamental problem in studying the latent-to-lytic switch of Epstein-Barr virus (EBV) and the viral lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction. Strategies to target EBV-positive tumors by inducing the viral lytic cycle with chemical agents are hindered by inefficient responses to stimuli. In vitro, even in the most susceptible cell lines, more than 50% of cells latently infected with EBV are refractory to induction of the lytic cycle. The mechanisms underlying the refractory state are not understood. We separated lytic from refractory Burkitt lymphoma-derived HH514-16 cells after treatment with an HDAC inhibitor, sodium butyrate. Both refractory- and lytic-cell populations responded to the inducing stimulus by hyperacetylation of histone H3. However, analysis of host cell gene expression showed that specific cellular transcripts Stat3, Fos, and interleukin-8 (IL-8) were preferentially upregulated in the refractory-cell population, while IL-6 was upregulated in the lytic population. STAT3 protein levels were also substantially increased in refractory cells relative to untreated or lytic cells. This increase in de novo expression resulted primarily in unphosphorylated STAT3. Examination of single cells revealed that high levels of STAT3 were strongly associated with the refractory state. The refractory state is manifest in a unique subpopulation of cells that exhibits different cellular responses than do lytic cells exposed to the same stimulus. Identifying characteristics of cells refractory to lytic induction relative to cells that undergo lytic activation will be an important step in developing a better understanding of the regulation of the EBV latent to lytic switch.

scholarly journals Genomic insights into the pathogenesis of Epstein–Barr virus-associated diffuse large B-cell lymphoma by whole-genome and targeted amplicon sequencing

2021 ◽  
Vol 11 (5) ◽  
Author(s):  
Niklas Gebauer ◽  
Axel Künstner ◽  
Julius Ketzer ◽  
Hanno M. Witte ◽  
Tobias Rausch ◽  
...  
Keyword(s):  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Amplicon Sequencing ◽  
Whole Genome ◽  
Barr Virus ◽  
Large B Cell Lymphoma ◽  
Epstein Barr ◽  
Large B Cell

AbstractEpstein–Barr virus (EBV)-associated diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS) constitute a distinct clinicopathological entity in the current World Health Organization (WHO) classification. However, its genomic features remain sparsely characterized. Here, we combine whole-genome sequencing (WGS), targeted amplicon sequencing (tNGS), and fluorescence in situ hybridization (FISH) from 47 EBV + DLBCL (NOS) cases to delineate the genomic landscape of this rare disease. Integrated WGS and tNGS analysis clearly distinguished this tumor type from EBV-negative DLBCL due to frequent mutations in ARID1A (45%), KMT2A/KMT2D (32/30%), ANKRD11 (32%), or NOTCH2 (32%). WGS uncovered structural aberrations including 6q deletions (5/8 patients), which were subsequently validated by FISH (14/32 cases). Expanding on previous reports, we identified recurrent alterations in CCR6 (15%), DAPK1 (15%), TNFRSF21 (13%), CCR7 (11%), and YY1 (6%). Lastly, functional annotation of the mutational landscape by sequential gene set enrichment and network propagation predicted an effect on the nuclear factor κB (NFκB) pathway (CSNK2A2, CARD10), IL6/JAK/STAT (SOCS1/3, STAT3), and WNT signaling (FRAT1, SFRP5) alongside aberrations in immunological processes, such as interferon response. This first comprehensive description of EBV + DLBCL (NOS) tumors substantiates the evidence of its pathobiological independence and helps stratify the molecular taxonomy of aggressive lymphomas in the effort for future therapeutic strategies.

A microassay for quantitatively detecting the epstein-barr virus receptor on single cells utilizing flow cytometry

1981 ◽  
Vol 3 (3) ◽  
pp. 127-136 ◽  
Author(s):  
Alan Wells ◽  
Harald Steen ◽  
Sem Saeland ◽  
Tore Godal ◽  
George Klein
Keyword(s):  
Flow Cytometry ◽  
Epstein Barr Virus ◽  
Single Cells ◽  
Virus Receptor ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals 4D Analyses Show That Replication Compartments Are Clonal Factories in Which Epstein–Barr Viral DNA Amplification Is Coordinated

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 140
Author(s):  
Thejaswi Nagaraju ◽  
Arthur Sugden ◽  
Bill Sugden
Keyword(s):  
Dna Synthesis ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Single Cells ◽  
Dna Amplification ◽  
Live Cell ◽  
Computational Simulations ◽  
Viral Dna ◽  
Barr Virus ◽  
Epstein Barr

Most DNA viruses must amplify their DNA to form new viral particles. To kickstart their DNA amplification, herpesviruses alter the host cell cycle dynamics by halting G1/S progression. Soon after, the viruses begin amplifying their DNA and halt any detectable cellular DNA synthesis. Viral DNA amplification takes place in specialized regions of the cell known as replication compartments. The genesis and maturation of replication compartments are not well understood. While replication compartments can only be visualized via microscopy, examining DNA synthetic events requires ensemble approaches. We have therefore exploited single-cell assays, including live-cell imaging, fluorescence in situ hybridization (FISH), and EdU-pulse labeling, in combination with computational simulations and ensemble approaches, to study the role of replication compartments in the DNA amplification of the Epstein–Barr virus (EBV). FISH revealed that each replication compartment initially contained a single DNA molecule which did not travel between compartments. DNA amplification lasted for 13–14 h in single cells, as shown by live cell imaging. Replication compartments eventually grew to occupy 30% of the nucleus, which itself grew by 50%. We found that early in the lytic phase, the availability of DNA templates limited DNA synthesis, while late in the lytic phase, the majority of viral DNA molecules no longer served as templates, which correlated with a drop in the levels of the replication protein. The eventual decline in DNA synthesis did not result from encapsidation; only 1–2% of the viral DNA was encapsidated. The levels of viral DNA synthesis in each compartment were similar. Therefore, the number of compartments determined the total amount of DNA synthesized and, consequently, the levels of amplified DNA. This finding was predicted by computational simulations of the amplification of the two distinct EBV derived replicons that we studied. Our results establish that replication compartments represent clonal factories for DNA amplification that are regulated coordinately during the lytic phase.

scholarly journals Long range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Nadja Brait ◽  
Büşra Külekçi ◽  
Irene Goerzer
Keyword(s):  
Gc Content ◽  
Amplicon Sequencing ◽  
Read Length ◽  
Third Generation Sequencing ◽  
Hcmv Strain ◽  
Novel Approach ◽  
Distinct Haplotype ◽  
Pcr Products ◽  
Long Range Pcr ◽  
Sequencing Platforms

Abstract Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

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