Epstein-Barr Virus Episome Physically Interacts with Active Regions of the Host Genome in Lymphoblastoid Cells

Wang, Luopin; Laing, Jun; Yan, Bingyu; Zhou, Hufeng; Ke, Liangru; Wang, Chong; Narita, Yohei; Zhang, Zonghao; Olson, Matthew R.; Afzali, Behdad; Zhao, Bo; Kazemian, Majid

doi:10.1128/jvi.01390-20

“…This is exemplified by the discovery of viral integration as a driver of oncogenesis in HPV-associated cancers 19 . We have previously used this approach to examine host-virus interactions during EBV infection and to determine how the EBV episome interacts with the human genome 4,5 . Even in the absence of infection, detailed analyses of RNA-seq data can deliver new insights into cell biology, including, for example, discovery of novel linear or circular genes and isoforms.…”

Section: Discussionmentioning

confidence: 99%

Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Yan

¹

,

Chakravorty

²

,

Mirabelli

³

et al. 2021

Preprint

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Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background noise. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

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“…This is exemplified by the discovery of viral integration as a driver of oncogenesis in HPVassociated cancers (3). We have previously deployed these methods to gain mechanistic insights into the pathophysiology of oncogenic viruses, such as Epstein-Barr virus (EBV), hepatitis B virus (HBV) and human papilloma virus (HPV) (1,(4)(5)(6). Even in the absence of infection, detailed analyses of RNA-seq data can deliver new insights into cell biology, including, for example, discovery of novel linear or circular genes and isoforms.…”

Section: Introductionmentioning

confidence: 99%

Host-Virus Chimeric Events in SARS-CoV-2-Infected Cells Are Infrequent and Artifactual

Yan

¹

,

Chakravorty

²

,

Mirabelli

³

et al. 2021

J Virol

Self Cite

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Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive-sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes. Importance The pathogenic mechanisms underlying SARS-CoV2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known why some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV2 infected cells and suggested that HVC events support potential “human genome invasion” and “integration” by SARS-CoV2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here we provide several evidences suggesting that the observed HVC events are likely artifactual.

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“…Moreover, genes are most often regulated by enhancers located within the same TAD. To study these chromatin interactions, the combination of multiple capture assays such as Hi-C, assay of transposase accessible chromatin sequencing (ATAC-seq) (Wang et al, 2020), whole-genome sequencing (WGS) (Adeel et al, 2021), RNA sequencing (RNA-seq) and/ or chromatin immunoprecipitation sequencing (ChIP-seq) (Zhang et al, 2020) has been developed. Up to date, several studies have characterized the structural variations of HPV-driven cervical cancer within certain integration hot spots (Cao et al, 2020), or by examining the genome-wide interactions using more unbiased approaches (Adeel et al, 2021;Groves et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, genes are most often regulated by enhancers located within the same TAD. To study these chromatin interactions, the combination of multiple capture assays such as Hi-C, assay of transposase accessible chromatin sequencing (ATAC-seq) ( Wang et al., 2020 ), whole-genome sequencing (WGS) ( Adeel et al., 2021 ), RNA sequencing (RNA-seq) and/or chromatin immunoprecipitation sequencing (ChIP-seq) ( Zhang et al., 2020 ) has been developed.…”

Section: Introductionmentioning

confidence: 99%

HPV16-LINC00393 Integration Alters Local 3D Genome Architecture in Cervical Cancer Cells

Xu

¹

,

Han

²

,

Ruan

³

et al. 2021

Front. Cell. Infect. Microbiol.

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High-risk human papillomavirus (hrHPV) infection and integration were considered as essential onset factors for the development of cervical cancer. However, the mechanism on how hrHPV integration influences the host genome structure remains not fully understood. In this study, we performed in situ high-throughput chromosome conformation capture (Hi-C) sequencing, chromatin immunoprecipitation and sequencing (ChIP-seq), and RNA-sequencing (RNA-seq) in two cervical cells, 1) NHEK normal human epidermal keratinocyte; and 2) HPV16-integrated SiHa tumorigenic cervical cancer cells. Our results reveal that the HPV-LINC00393 integrated chromosome 13 exhibited significant genomic variation and differential gene expression, which was verified by calibrated CTCF and H3K27ac ChIP-Seq chromatin restructuring. Importantly, HPV16 integration led to differential responses in topologically associated domain (TAD) boundaries, with a decrease in the tumor suppressor KLF12 expression downstream of LINC00393. Overall, this study provides significant insight into the understanding of HPV16 integration induced 3D structural changes and their contributions on tumorigenesis, which supplements the theory basis for the cervical carcinogenic mechanism of HPV16 integration.

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Epstein-Barr Virus Episome Physically Interacts with Active Regions of the Host Genome in Lymphoblastoid Cells

Cited by 28 publications

References 51 publications

Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Host-Virus Chimeric Events in SARS-CoV-2-Infected Cells Are Infrequent and Artifactual

HPV16-LINC00393 Integration Alters Local 3D Genome Architecture in Cervical Cancer Cells

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