| 1. |
- Holmqvist, Isak, et al.
(författare)
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FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
- 2021
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Ingår i: Rna. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:10, s. 1127-1139
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Tidskriftsartikel (refereegranskat)abstract
- Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this work-flow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
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| 2. |
- Hussein, Brwa Ali, 1984, et al.
(författare)
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NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells
- 2023
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Ingår i: JOURNAL FOR IMMUNOTHERAPY OF CANCER. - 2051-1426. ; 11:8
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Tidskriftsartikel (refereegranskat)abstract
- Background The natural killer (NK) complex (NKC) harbors multiple genes such as KLRC1 (encoding NKG2A) and KLRK1 (encoding NKG2D) that are central to regulation of NK cell function. We aimed at determining to what extent NKC haplotypes impact on NK cell repertoire and function, and whether such gene variants impact on outcome of IL-2-based immunotherapy in acute myeloid leukemia (AML).Methods Genotype status of NKG2D rs1049174 and NKG2A rs1983526 was determined using the TaqMan-Allelic discrimination approach. To dissect the impact of single nucloetide polymorphim (SNP) on NK cell function, we engineered the K562 cell line with CRISPR to be killed in a highly NKG2D-dependent fashion. NK cells were assayed for degranulation, intracellular cytokine production and cytotoxicity using flow cytometry.Results In AML patients receiving immunotherapy, the NKG2A gene variant, rs1983526, was associated with superior leukemia-free survival and overall survival. We observed that superior NK degranulation from individuals with the high-cytotoxicity NKG2D variant was explained by presence of a larger, highly responsive NKG2A(+) subset. Notably, NK cells from donors homozygous for a favorable allele encoding NKG2A mounted stronger cytokine responses when challenged with leukemic cells, and NK cells from AML patients with this genotype displayed higher accumulation of granzyme B during histamine dihydrochloride/IL-2 immunotherapy. Additionally, among AML patients, the NKG2A SNP defined a subset of patients with HLA-B-21 TT with a strikingly favorable outcome.Conclusions The study results imply that a dimorphism in the NKG2A gene is associated with enhanced NK cell effector function and improved outcome of IL-2-based immunotherapy in AML.
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| 3. |
- Kristenson, Linnea, 1991, et al.
(författare)
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Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity
- 2024
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Ingår i: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - 0027-8424 .- 1091-6490. ; 121:15
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Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-gamma signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7 - H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta- subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock - out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM - 3, restored the NK cell ability to eliminate TMEM30A- mutated cells. The key role of the TIM - 3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM - 3 in the realm of cancer immunotherapy.
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| 4. |
- Olausson, Josefin, 1983, et al.
(författare)
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Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Infection in the central nervous system is a severe condition associated with high morbidity and mortality. Despite ample testing, the majority of encephalitis and meningitis cases remain undiagnosed. Metagenomic sequencing of cerebrospinal fluid has emerged as an unbiased approach to identify rare microbes and novel pathogens. However, several major hurdles remain, including establishment of individual limits of detection, removal of false positives and implementation of universal controls. Twenty-one cerebrospinal fluid samples, in which a known pathogen had been positively identified by available clinical techniques, were subjected to metagenomic DNA sequencing. Fourteen samples contained minute levels of Epstein-Barr virus. The detection threshold for each sample was calculated by using the total leukocyte content in the sample and environmental contaminants found in the bioinformatic classifiers. Virus sequences were detected in all ten samples, in which more than one read was expected according to the calculations. Conversely, no viral reads were detected in seven out of eight samples, in which less than one read was expected according to the calculations. False positive pathogens of computational or environmental origin were readily identified, by using a commonly available cell control. For bacteria, additional filters including a comparison between classifiers removed the remaining false positives and alleviated pathogen identification. Here we show a generalizable method for identification of pathogen species using DNA metagenomic sequencing. The choice of bioinformatic method mainly affected the efficiency of pathogen identification, but not the sensitivity of detection. Identification of pathogens requires multiple filtering steps including read distribution, sequence diversity and complementary verification of pathogen reads.
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| 5. |
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| 6. |
- Muylaert, Isabella, 1965, et al.
(författare)
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Replication and Recombination of Herpes Simplex Virus DNA
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 286:18
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Forskningsöversikt (refereegranskat)abstract
- Replication of Herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of viral proteins: the UL30/42 DNA polymerase, the UL5,8,52 helicase-primase and the UL29 single-strand DNA binding protein ICP8. The replisome is loaded on origins of replication by an initiator protein OBP/UL9. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here we review significant new developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor and the single-strand DNA binding protein, the reconstitution of a functional replisome in vitro, the elucidation of the mechanism for activation of origins of DNA replication, the identification of cellular proteins actively involved in or responding to viral DNA replication and, finally, the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.
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| 7. |
- Nordén, Rickard, 1977, et al.
(författare)
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Quantification of torque teno virus and Epstein-Barr virus is of limited value for predicting the net state of immunosuppression after lung transplantation
- 2018
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Ingår i: Open Forum Infectious Diseases. - : Oxford University Press (OUP). - 2328-8957. ; 5:4
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Tidskriftsartikel (refereegranskat)abstract
- Background. Major hurdles for survival after lung transplantation are rejections and infectious complications. Adequate methods for monitoring immune suppression status are lacking. Here, we evaluated quantification of torque teno virus (TTV) and Epstein-Barr virus (EBV) as biomarkers for defining the net state of immunosuppression in lung-transplanted patients. Methods. This prospective single-center study included 98 patients followed for 2 years after transplantation. Bacterial infections, fungal infections, viral respiratory infections (VRTI), cytomegalovirus (CMV) viremia, and acute rejections, as well as TTV and EBV levels, were monitored. Results. The levels of torque teno virus DNA increased rapidly after transplantation, likely due to immunosuppressive treatment. A modest increase in levels of Epstein-Barr virus DNA was also observed after transplantation. There were no associations between either TTV or EBV and infectious events or acute rejection, respectively, during follow-up. When Tacrolimus was the main immunosuppressive treatment, TTV DNA levels were significantly elevated 6-24 months after transplantation as compared with Cyclosporine treatment. Conclusions. Although replication of TTV, but not EBV, appears to reflect the functionality of the immune system, depending on the type of immunosuppressive treatment, quantification of TTV or EBV as biomarkers has limited potential for defining the net state of immune suppression.
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| 8. |
- Norder, Helene, et al.
(författare)
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High Frequency of Either Altered Pre-Core StartCodon or Weakened Kozak Sequence in the CorePromoter Region in Hepatitis B Virus A1 Strainsfrom Rwanda.
- 2019
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Ingår i: Genes. - : MDPI AG. - 2073-4425. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus (HBV) is endemic in Rwanda and is a major etiologic agent for chronic liver disease in the country. In a previous analysis of HBV strains from Rwanda, the S genes of most strains segregated into one single clade of subgenotype, A1. More than half (55%) of the anti-HBe positive individuals were viremic. In this study, 23 complete HBV genomes and the core promoter region (CP) from 18 additional strains were sequenced. Phylogenetic analysis of complete genomes confirmed that most Rwandan strain formed a single unique clade, within subgenotype A1. Strains from 17 of 22 (77%) anti-HBe positive HBV carriers had either mutated the precore start codon (9 strains with either CUG, ACG, UUG, or AAG) or mutations in the Kozak sequence preceding the pre-core start codon (8 strains). These mutually exclusive mutations were also identified in subgenotypes A1 (70/266; 26%), A2 (12/255; 5%), and A3 (26/49; 53%) sequences from the GenBank. The results showed that previous, rarely described HBV variants, expressing little or no HBeAg, are selected in anti-HBe positive subgenotype Al carriers from Rwanda and that mutations reducing HBeAg synthesis might be unique for a particular HBV clade, not just for a specific genotype or subgenotype.
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| 9. |
- Nyström, Kristina, 1977, et al.
(författare)
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Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus
- 2018
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 92:19
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Tidskriftsartikel (refereegranskat)abstract
- A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies. IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.
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| 10. |
- Nyström, Kristina, 1977, et al.
(författare)
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Ribavirin: Pharmacology, multiple modes of action and possible future perspectives
- 2019
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Ingår i: Future Virology. - : Future Medicine Ltd. - 1746-0794 .- 1746-0808. ; 14, s. 153-160
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Forskningsöversikt (refereegranskat)abstract
- © 2019 2019 Martin Lagging. Ribavirin is a unique guanosine analog with broad-spectrum activity against many RNA and DNA viruses. In addition to its mutational properties, ribavirin exerts extensive perturbation of cellular and viral gene expression. Furthermore, recent advances indicate that the impact of ribavirin on divergent cellular and viral pathways may be concentration dependent. This review aims at providing an overview of the pharmacology and multiple modes of action of ribavirin as well as pointing to possible novel future uses.
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| 11. |
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| 12. |
- Ringlander, Johan, et al.
(författare)
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Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA
- 2020
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Ingår i: Journal of Viral Hepatitis. - : Wiley. - 1352-0504 .- 1365-2893. ; 27:11, s. 1162-1170
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3 ' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.
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| 13. |
- Tang, Ka-Wei, 1983, et al.
(författare)
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Absence of cytomegalovirus in high-coverage DNA sequencing of human glioblastoma multiforme.
- 2015
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Ingår i: International journal of cancer. Journal international du cancer. - : Wiley. - 1097-0215. ; 136:4, s. 977-981
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Tidskriftsartikel (refereegranskat)abstract
- Cytomegalovirus (CMV) has been proposed to be associated with glioblastoma multiforme, but there are conflicting results including lack of CMV mRNA in transcriptome sequencing data. Here, we utilized deep-coverage whole-genome sequencing data to detect latent CMV DNA in surgically resected tumors and to assess the relative proportions of viral and human DNA. We did not find traces of CMV in 52.6 billion DNA sequencing reads from 34 glioblastomas. By statistical analysis, we conclude that should the virus be present in these tumors, the average CMV level does not exceed one virus per 240,000 tumor cells (99% CI).
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| 14. |
- Tang, Ka-Wei, 1983
(författare)
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Herpes Simplex Virus 1 DNA replication and its role in recombination and transcription
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Herpes simplex virus 1 (HSV-1) is one of nine different herpesvirus infecting man. They are all capable of establishing a life-long latent state following the primary infection. HSV-1 as well as other herpesviruses may reactivate from the latent state and give rise to a productive infection with clinical symptoms or asymptomatic shedding. HSV-1 infections are primarily treated by targeting the viral DNA replication carried out by a molecular machinery, a replisome, encoded by the virus. Here we examine the mechanism of initiation of viral DNA replication and also how viral DNA replication interacts with DNA recombination and gene expression. In our first study we examined the initiation-step of HSV-1 replication. The origin binding protein (OBP) initiates replication by binding to the origin of replication (oriS and/or oriL). We showed, using phylogenetics and biochemical experiments, that there was a step-wise evolutionary development of herpesvirus DNA replication initiation. The initial divergence was seen in herpesviruses acquiring an amino-acid motif RVKNL in OBP which binds the sequence TTCGCAC in the oriS. The next step was the development of an ICP8 binding motif at the C-terminus of OBP and finally the arrangement of the binding-sites for OBP in the oriS-sequence and the ability to form a stable hairpin. We presented molecular in vitro data to support the phylogenetic analysis and thereby defining essential motifs in OBP for protein-protein and protein-DNA interactions. The next study was focused on genetic recombination between different HSV-1 strains. We followed the propagation of HSV-1 in cells infected with one to three genotypes of HSV-1 and calculated the number of recombination events. We found evidence for Rad51 and Rad52 involvement in recombination of the unique long and unique short gene segments of HSV-1. We also observed an increased recombination rate in cells with retarded ligation of Okazaki-fragments. The fidelity of recombination in virus propagated through mixed infections appears to be high since expansion or shortening of repeated sequences in the US7 gene was not detected. In the third study we examined the replication-coupled transcription of HSV-1 late genes, which are known to depend on DNA replication for efficient expression. Using chromatin immuno-precipitation we could determine that recruitment of RNA polymerase II to late gene promoters occur even in the absence of replication. Recruitment was dependent on ICP4, but delayed in comparison with early gene promoters. These observations suggested the involvement of transcription elongation and/or maturation in the expression of gamma genes. By using the drug DRB, which inhibits the kinase CDK9, a component of the positive transcription elongation factor B, and siRNA against Spt5, a transcription processivity factor, we could show a specific impairment of late gene expression, with only minimal effect on early gene expression and DNA synthesis. We suggest that CDK9 and Spt5 are specifically recruited to replicated late genes and mediate a maturation of transcribed mRNA for nuclear exit. In summary, we have studied essential molecular processes in the HSV-1 life cycle and identified molecular interactions as well as mechanistic pathways, which may serve as future drug targets.
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| 15. |
- Tang, Ka-Wei, 1983, et al.
(författare)
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Rad51 and rad52 are involved in homologous recombination of replicating herpes simplex virus DNA.
- 2014
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 9:11
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Tidskriftsartikel (refereegranskat)abstract
- Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.
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| 16. |
- Tang, Ka-Wei, 1983, et al.
(författare)
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The landscape of viral expression and host gene fusion and adaptation in human cancer
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Viruses cause 10-15% of all human cancers. Massively parallel sequencing has recently proved effective for uncovering novel viruses and virus-tumour associations, but this approach has not yet been applied to comprehensive patient cohorts. Here we screen a diverse landscape of human cancer, encompassing 4,433 tumours and 19 cancer types, for known and novel expressed viruses based on >700 billion transcriptome sequencing reads from The Cancer Genome Atlas Research Network. The resulting map confirms and extends current knowledge. We observe recurrent fusion events, including human papillomavirus insertions in RAD51B and ERBB2. Patterns of coadaptation between host and viral gene expression give clues to papillomavirus oncogene function. Importantly, our analysis argues strongly against viral aetiology in several cancers where this has frequently been proposed. We provide a virus-tumour map of unprecedented scale that constitutes a reference for future studies of tumour-associated viruses using transcriptome sequencing data.
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| 17. |
- Tang, Ka-Wei, 1983, et al.
(författare)
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Tumour virology in the era of high-throughput genomics
- 2017
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Ingår i: Philosophical Transactions of the Royal Society B-Biological Sciences. - : The Royal Society. - 0962-8436 .- 1471-2970. ; 372:1732
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Tidskriftsartikel (refereegranskat)abstract
- With the advent of massively parallel sequencing, oncogenic viruses in tumours can now be detected in an unbiased and comprehensive manner. Additionally, new viruses or strains can be discovered based on sequence similarity with known viruses. Using this approach, the causative agent for Merkel cell carcinoma was identified. Subsequent studies using data from large collections of tumours have confirmed models built during decades of hypothesis-driven and low-throughput research, and a more detailed and comprehensive description of virus-tumour associations have emerged. Notably, large cohorts and high sequencing depth, in combination with newly developed bioinformatical techniques, have made it possible to rule out several suggested virus-tumour associations with a high degree of confidence. In this review we discuss possibilities, limitations and insights gained from using massively parallel sequencing to characterize tumours with viral content, with emphasis on detection of viral sequences and genomic integration events.
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| 18. |
- Zhao, Zhiyuan, et al.
(författare)
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CDK9 and SPT5 proteins are specifically required for expression of herpes simplex virus 1 replication-dependent late genes
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 292:37, s. 15489-15500
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Tidskriftsartikel (refereegranskat)abstract
- DNA replication greatly enhances expression of the herpes simplex virus 1 (HSV-1) gamma 2 late genes by still unknown mechanisms. Here, we demonstrate that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK9, suppresses expression of gamma 2 late genes with an IC50 of 5 mu M, which is at least 10 times lower than the IC50 value required for inhibition of expression of early genes. The effect of DRB could not be explained by inhibition of DNA replication per se or loading of RNA polymerase II to late promoters and subsequent reduction of transcription. Instead, DRB reduces accumulation of gamma 2 late mRNA in the cytoplasm. In addition, we show that siRNA-mediated knockdown of the transcription factor SPT5, but not NELF-E, also gives rise to a specific inhibition of HSV-1 late gene expression. Finally, addition of DRB reduces co-immunoprecipitation of ICP27 using an anti-SPT5 antibody. Our results suggest that efficient expression of replication-dependent gamma 2 late genes is, at least in part, regulated by CDK9 dependent co-and/or post-transcriptional events involving SPT5 and ICP27.
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| 19. |
- Ziegler, P., et al.
(författare)
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A primary nasopharyngeal three-dimensional air-liquid interface cell culture model of the pseudostratified epithelium reveals differential donor- and cell type-specific susceptibility to Epstein-Barr virus infection
- 2021
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Ingår i: Plos Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:4
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, alpha-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (similar to 0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC. Author summary It has been known for over 50 years that EBV latent infection is associated with NPC. Despite many advances from studies in 2-dimensional cell culture, many aspects of EBV molecular pathogenesis in the nasopharynx remain undefined because the cell types and the biology of the nasopharyngeal epithelium can only be faithfully captured in 3-dimensional cell culture. In the stratified epithelium, cellular differentiation triggers lytic infection but it is not clear to what degree the pseudostratified epithelium is involved. The pseudostratified epithelium is abundant in the lateral wall where the lymphoid-rich fossa of Rosenmuller is located and is a site where NPC tumors most often arises. While the oral epithelium is a site of EBV replication, whether the nasopharyngeal epithelium is a major source of EBV shedding in the nasopharynx is not well defined. Here, we present a 3-dimensional organoid model of the nasopharyngeal pseudostratified epithelium showing that such cells can be infected with EBV in some donor cultures, with examples of both latent and lytic infection. We propose that the cell types of the pseudostratified epithelium should be considered a component of EBV pathogenesis in the nasopharynx and that the difference in donor susceptibility and latent/lytic infection could influence EBV's fitness in the nasopharynx.
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