Search

Simple Summary: This research aimed to identify, through an in silico approach using interaction networks, a potential biomarker associated with HPV 16 integration in cervical cancer. MAGI-1 was selected, and the expression profiles and changes in subcellular localization in cell lines and samples were evaluated in patients with premalignant lesions and invasive squamous carcinoma with HPV 16 in an integrated state. MAGI-1 is a potential biomarker that differentiates a normal cell with integrated HPV 16 from early cervical lesions. HPV 16 integration is crucial for the onset and progression of premalignant lesions to invasive squamous cell carcinoma (ISCC) because it promotes the amplification of proto-oncogenes and the silencing of tumor suppressor genes; some of these are proteins with PDZ domains involved in homeostasis and cell polarity. Through a bioinformatics approach based on interaction networks, a group of proteins associated with HPV 16 infection, PDZ domains, and direct physical interaction with E6 and related to different hallmarks of cancer were identified. MAGI-1 was selected to evaluate the expression profile and subcellular localization changes in premalignant lesions and ISCC with HPV 16 in an integrated state in cervical cytology; the profile expression of MAGI-1 diminished according to lesion grade. Surprisingly, in cell lines CaSki and SiHa, the protein localization was cytoplasmic and nuclear. In contrast, in histological samples, a change in subcellular localization from the cytoplasm in low-grade squamous intraepithelial lesions (LSIL) to the nucleus in the high-grade squamous intraepithelial lesion (HSIL) was observed; in in situ carcinomas and ISCC, MAGI-1 expression was absent. In conclusion, MAGI-1 expression could be a potential biomarker for distinguishing those cells with normal morphology but with HPV 16 integrated from those showing morphology-related uterine cervical lesions associated with tumor progression. [ABSTRACT FROM AUTHOR]

The site‐specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage‐encoded Int and Xis proteins and the host‐encoded integration host factor and factor for inversion stimulation proteins. Structures of higher‐order protein–DNA complexes of the four‐way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site‐specific recombination, with an emphasis on key studies completed over the last decade. [ABSTRACT FROM AUTHOR]

The clearance or transcriptional silencing of integrated HBV DNA is crucial for achieving a functional cure in patients with chronic hepatitis B and reducing the risk of hepatocellular carcinoma development. The PLC/PRF/5 cell line is commonly used as an in vitro model for studying HBV integration. In this study, we employed a range of multi‐omics techniques to gain a panoramic understanding of the characteristics of HBV integration in PLC/PRF/5 cells and to reveal the transcriptional regulatory mechanisms of integrated HBV DNA. Transcriptome long‐read sequencing (ONT) was conducted to analyze and characterize the transcriptional activity of different HBV DNA integration sites in PLC/PRF/5 cells. Additionally, we collected data related to epigenetic regulation, including whole‐genome bisulfite sequencing (WGBS), histone chromatin immunoprecipitation sequencing (ChIP‐seq), and assays for transposase‐accessible chromatin using sequencing (ATAC‐seq), to explore the potential mechanisms involved in the transcriptional regulation of integrated HBV DNA. Long‐read RNA sequencing analysis revealed significant transcriptional differences at various integration sites in the PLC/PRF/5 cell line, with higher HBV DNA transcription levels at integration sites on chr11, chr13, and the chr13/chr5 fusion chromosome t (13:5). Combining long‐read DNA and RNA sequencing results, we found that transcription of integrated HBV DNA generally starts downstream of the SP1, SP2, or XP promoters. ATAC‐seq data confirmed that chromatin accessibility has limited influence on the transcription of integrated HBV DNA in the PLC/PRF/5 cell line. Analysis of WGBS data showed that the methylation intensity of integrated HBV DNA was highly negatively correlated with its transcription level (r = −0.8929, p = 0.0123). After AzaD treatment, the transcription level of integrated HBV DNA significantly increased, especially for the integration chr17, which had the highest level of methylation. Through ChIP‐seq data, we observed the association between histone modification of H3K4me3 and H3K9me3 with the transcription of integrated HBV DNA. Our findings suggest that the SP1, SP2 and XP in integrated HBV DNA, methylation level of surrounding host chromosome, and histone modifications affect the transcription of integrated HBV DNA in PLC/PRF/5 cells. This provides important clues for future studies on the expression and regulatory mechanisms of integrated HBV. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well‐studied investigation about virus–host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins. Materials and methods: High‐throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing. Results: HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD‐L1 expression demonstrated increased integration sites (p =.024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty‐nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process. Conclusions: HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus–host interactions, enhancing our understanding of HPV‐related carcinogenesis mechanisms. [ABSTRACT FROM AUTHOR]

Persistent high-risk human papillomavirus (HPV) infection is a pivotal factor in the progression of cervical cancer. In recent years, an increasing interest has emerged in comprehending the influence of HPV on head and neck squamous cell carcinoma (HNSCC). Notably, it is well established that HPV-associated HNSCC show cases with distinct molecular and clinical attributes compared to HPV-negative cases. The present study delves into the epigenetic landscape of HPV16, specifically its L1 gene and untranslated region (UTR), through pyrosequencing, while the HPV16 DNA physical status was evaluated using E2/E6 ratio analysis in HPV16-positive HNSCC FFPE biopsies. Our findings reveal substantial methylation across six sites within the HPV16 L1 gene and seven sites in the UTR. Specifically, methylation percentages of two L1 CpG sites (7136, 7145) exhibit significant associations with tumor histological grade (p < 0.01), while proving concurrent methylation across multiple sites. The HPV16 DNA physical status was not correlated with the methylation of viral genome or tumor characteristics. This is the first study that examines epigenetic modifications and the HPV16 DNA physical status in Greek HNSCC patients. Our findings suggest an orchestrated epigenetic modulation among specific sites, impacting viral gene expression and intricate virus–host interactions. [ABSTRACT FROM AUTHOR]

Chronic infection of hepatitis B virus (HBV) is the major cause of hepatocellular carcinoma (HCC). Notably, 90% of HBV‐positive HCC cases exhibit detectable HBV integrations, hinting at the potential early entanglement of these viral integrations in tumorigenesis and their subsequent oncogenic implications. Nevertheless, the precise chronology of integration events during HCC tumorigenesis, alongside their sequential structural patterns, has remained elusive thus far. In this study, we applied whole‐genome sequencing to multiple biopsies extracted from six HBV‐positive HCC cases. Through this approach, we identified point mutations and viral integrations, offering a blueprint for the intricate tumor phylogeny of these samples. The emergent narrative paints a rich tapestry of diverse evolutionary trajectories characterizing the analyzed tumors. We uncovered oncogenic integration events in some samples that appear to happen before and during the initiation stage of tumor development based on their locations in reconstituted trajectories. Furthermore, we conducted additional long‐read sequencing of selected samples and unveiled integration‐bridged chromosome rearrangements and tandem repeats of the HBV sequence within integrations. In summary, this study revealed premalignant oncogenic and sequential complex integrations and highlighted the contributions of HBV integrations to HCC development and genome instability. [ABSTRACT FROM AUTHOR]

Abstract Background Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well‐studied investigation about virus–host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins. Materials and methods High‐throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing. Results HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD‐L1 expression demonstrated increased integration sites (p = .024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty‐nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process. Conclusions HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus–host interactions, enhancing our understanding of HPV‐related carcinogenesis mechanisms.

HPV 16 integration is crucial for the onset and progression of premalignant lesions to invasive squamous cell carcinoma (ISCC) because it promotes the amplification of proto-oncogenes and the silencing of tumor suppressor genes; some of these are proteins with PDZ domains involved in homeostasis and cell polarity. Through a bioinformatics approach based on interaction networks, a group of proteins associated with HPV 16 infection, PDZ domains, and direct physical interaction with E6 and related to different hallmarks of cancer were identified. MAGI-1 was selected to evaluate the expression profile and subcellular localization changes in premalignant lesions and ISCC with HPV 16 in an integrated state in cervical cytology; the profile expression of MAGI-1 diminished according to lesion grade. Surprisingly, in cell lines CaSki and SiHa, the protein localization was cytoplasmic and nuclear. In contrast, in histological samples, a change in subcellular localization from the cytoplasm in low-grade squamous intraepithelial lesions (LSIL) to the nucleus in the high-grade squamous intraepithelial lesion (HSIL) was observed; in in situ carcinomas and ISCC, MAGI-1 expression was absent. In conclusion, MAGI-1 expression could be a potential biomarker for distinguishing those cells with normal morphology but with HPV 16 integrated from those showing morphology-related uterine cervical lesions associated with tumor progression.

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies.

Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for in vivo and ex vivo delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.

The link between viruses and cancer has intrigued scientists for decades. Certain viruses have been shown to be vital in the development of various cancers by integrating viral DNA into the host genome and activating viral oncogenes. These viruses include the Human Papillomavirus (HPV), Hepatitis B and C Viruses (HBV and HCV), Epstein-Barr Virus (EBV), and Human T-Cell Leukemia Virus (HTLV-1), which are all linked to the development of a myriad of human cancers. Third-generation sequencing technologies have revolutionized our ability to study viral integration events at unprecedented resolution in recent years. They offer long sequencing capabilities along with the ability to map viral integration sites, assess host gene expression, and track clonal evolution in cancer cells. Recently, researchers have been exploring the application of Oxford Nanopore Technologies (ONT) nanopore sequencing and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) sequencing in cancer research. As viral integration is crucial to the development of cancer via viruses, third-generation sequencing would provide a novel approach to studying the relationship interlinking viral oncogenes, viruses, and cancer. This review article explores the molecular mechanisms underlying viral oncogenesis, the role of viruses in cancer development, and the impact of third-generation sequencing on our understanding of viral integration into the human genome.

Background: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. Results: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. Conclusion: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses. [ABSTRACT FROM AUTHOR]

Simple Summary: Cells infected with high-risk human papillomaviruses (HPV) can accumulate DNA damage and eventually transform into HPV-driven cancers. Genome instability, or the progressive accumulation of DNA alterations (e.g., mutations), in HPV-infected cells is directly induced by the HPV genes and indirectly promoted by HPV infection through the consequences of chronic infection maintenance, increased cell growth, and accumulation of damaging mutations in genes that themselves affect genome instability. While the HPV genome typically exists as a separate entity within cells, genome instability increases the chances of HPV integrating within the host (human) genome, which is common in HPV-induced cancers. The DNA regions surrounding HPV integrations are unstable and can undergo complex alterations that affect both human and HPV genes. This review discusses HPV-dependent and -independent drivers and mechanisms of genome instability in HPV-driven cancers, both globally and around sites of HPV integration, and describes the changes induced in the tumour genome. Human papillomavirus (HPV) is the causative driver of cervical cancer and a contributing risk factor of head and neck cancer and several anogenital cancers. HPV's ability to induce genome instability contributes to its oncogenicity. HPV genes can induce genome instability in several ways, including modulating the cell cycle to favour proliferation, interacting with DNA damage repair pathways to bring high-fidelity repair pathways to viral episomes and away from the host genome, inducing DNA-damaging oxidative stress, and altering the length of telomeres. In addition, the presence of a chronic viral infection can lead to immune responses that also cause genome instability of the infected tissue. The HPV genome can become integrated into the host genome during HPV-induced tumorigenesis. Viral integration requires double-stranded breaks on the DNA; therefore, regions around the integration event are prone to structural alterations and themselves are targets of genome instability. In this review, we present the mechanisms by which HPV-dependent and -independent genome instability is initiated and maintained in HPV-driven cancers, both across the genome and at regions of HPV integration. [ABSTRACT FROM AUTHOR]

Persistent high-risk human papillomavirus (HPV) infection is a pivotal factor in the progression of cervical cancer. In recent years, an increasing interest has emerged in comprehending the influence of HPV on head and neck squamous cell carcinoma (HNSCC). Notably, it is well established that HPV-associated HNSCC show cases with distinct molecular and clinical attributes compared to HPV-negative cases. The present study delves into the epigenetic landscape of HPV16, specifically its L1 gene and untranslated region (UTR), through pyrosequencing, while the HPV16 DNA physical status was evaluated using E2/E6 ratio analysis in HPV16-positive HNSCC FFPE biopsies. Our findings reveal substantial methylation across six sites within the HPV16 L1 gene and seven sites in the UTR. Specifically, methylation percentages of two L1 CpG sites (7136, 7145) exhibit significant associations with tumor histological grade (p < 0.01), while proving concurrent methylation across multiple sites. The HPV16 DNA physical status was not correlated with the methylation of viral genome or tumor characteristics. This is the first study that examines epigenetic modifications and the HPV16 DNA physical status in Greek HNSCC patients. Our findings suggest an orchestrated epigenetic modulation among specific sites, impacting viral gene expression and intricate virus–host interactions.

In October 2021, 59 scientists from 14 countries and 13 U.S. states collaborated virtually in the Third Annual Baylor College of Medicine & DNANexus Structural Variation hackathon. The goal of the hackathon was to advance research on structural variants (SVs) by prototyping and iterating on open-source software. This led to nine hackathon projects focused on diverse genomics research interests, including various SV discovery and genotyping methods, SV sequence reconstruction, and clinically relevant structural variation, including SARS-CoV-2 variants. Repositories for the projects that participated in the hackathon are available at https://github.com/collaborativebioinformatics.

A typical characteristics of polydnavirus (PDV) infection is a persistent immunosuppression, governed by the viral integration and expression of virulence genes. Recently, activation of caspase-3 by Microplitis bicoloratus bracovirus (MbBV) to cleave Innexins, gap junction proteins, has been highlighted, further promoting apoptotic cell disassembly and apoptotic body (AB) formation. However, whether ABs play a role in immune suppression remains to be determined. Herein, we show that ABs transmitted immunosuppressive signaling, causing recipient cells to undergo apoptosis and dismigration. Furthermore, the insertion of viral–host integrated motif sites damaged the host genome, stimulating eIF5A nucleocytoplasmic transport and activating the eIF5A-hypusination translation pathway. This pathway specifically translates apoptosis-related host proteins, such as P53, CypA, CypD, and CypJ, to drive cellular apoptosis owing to broken dsDNA. Furthermore, translated viral proteins, such Vank86, 92, and 101, known to complex with transcription factor Dip3, positively regulated DHYS and DOHH transcription maintaining the activation of the eIF5A-hypusination. Mechanistically, MbBV-mediated extracellular vesicles contained inserted viral fragments that re-integrated into recipients, potentially via the homologous recombinant repair system. Meanwhile, this stimulation regulated activated caspase-3 levels via PI3K/AKT 308 and 473 dephosphorylation to promote apoptosis of granulocyte-like recipients Sf9 cell; maintaining PI3K/AKT 473 phosphorylation and 308 dephosphorylation inhibited caspase-3 activation leading to dismigration of plasmatocyte-like recipient High Five cells. Together, our results suggest that integration-mediated eIF5A hypusination drives extracellular vesicles for continuous immunosuppression. [ABSTRACT FROM AUTHOR]

A typical characteristics of polydnavirus (PDV) infection is a persistent immunosuppression, governed by the viral integration and expression of virulence genes. Recently, activation of caspase-3 by Microplitis bicoloratus bracovirus (MbBV) to cleave Innexins, gap junction proteins, has been highlighted, further promoting apoptotic cell disassembly and apoptotic body (AB) formation. However, whether ABs play a role in immune suppression remains to be determined. Herein, we show that ABs transmitted immunosuppressive signaling, causing recipient cells to undergo apoptosis and dismigration. Furthermore, the insertion of viral–host integrated motif sites damaged the host genome, stimulating eIF5A nucleocytoplasmic transport and activating the eIF5A-hypusination translation pathway. This pathway specifically translates apoptosis-related host proteins, such as P53, CypA, CypD, and CypJ, to drive cellular apoptosis owing to broken dsDNA. Furthermore, translated viral proteins, such Vank86, 92, and 101, known to complex with transcription factor Dip3, positively regulated DHYS and DOHH transcription maintaining the activation of the eIF5A-hypusination. Mechanistically, MbBV-mediated extracellular vesicles contained inserted viral fragments that re-integrated into recipients, potentially via the homologous recombinant repair system. Meanwhile, this stimulation regulated activated caspase-3 levels via PI3K/AKT 308 and 473 dephosphorylation to promote apoptosis of granulocyte-like recipients Sf9 cell; maintaining PI3K/AKT 473 phosphorylation and 308 dephosphorylation inhibited caspase-3 activation leading to dismigration of plasmatocyte-like recipient High Five cells. Together, our results suggest that integration-mediated eIF5A hypusination drives extracellular vesicles for continuous immunosuppression.

Abstract Background Several bioinformatics pipelines have been developed to detect sequences from viruses that integrate into the human genome because of the health relevance of these integrations, such as in the persistence of viral infection and/or in generating genotoxic effects, often progressing into cancer. Recent genomics and metagenomics analyses have shown that viruses also integrate into the genome of non-model organisms (i.e., arthropods, fish, plants, vertebrates). However, rarely studies of endogenous viral elements (EVEs) in non-model organisms have gone beyond their characterization from reference genome assemblies. In non-model organisms, we lack a thorough understanding of the widespread occurrence of EVEs and their biological relevance, apart from sporadic cases which nevertheless point to significant roles of EVEs in immunity and regulation of expression. The concomitance of repetitive DNA, duplications and/or assembly fragmentations in a genome sequence and intrasample variability in whole-genome sequencing (WGS) data could determine misalignments when mapping data to a genome assembly. This phenomenon hinders our ability to properly identify integration sites. Results To fill this gap, we developed ViR, a pipeline which solves the dispersion of reads due to intrasample variability in sequencing data from both single and pooled DNA samples thus ameliorating the detection of integration sites. We tested ViR to work with both in silico and real sequencing data from a non-model organism, the arboviral vector Aedes albopictus. Potential viral integrations predicted by ViR were molecularly validated supporting the accuracy of ViR results. Conclusion ViR will open new venues to explore the biology of EVEs, especially in non-model organisms. Importantly, while we generated ViR with the identification of EVEs in mind, its application can be extended to detect any lateral transfer event providing an ad-hoc sequence to interrogate.

The identification of structural variations (SVs) and viral integrations in circulating tumor DNA (ctDNA) is a key step in precision oncology that may assist clinicians in treatment selection and monitoring. However, due to the short fragment size of ctDNA, it is challenging to accurately detect low-frequency SVs or SVs involving complex junctions in ctDNA sequencing data. Here, we describe Aperture, a new fast SV caller that applies a unique strategy of |$k$| -mer-based searching, binary label–based breakpoint detection and candidate clustering to detect SVs and viral integrations with high sensitivity, especially when junctions span repetitive regions. Aperture also employs a barcode-based filter to ensure specificity. Compared with existing methods, Aperture exhibits superior sensitivity and specificity in simulated, reference and real data tests, especially at low dilutions. Additionally, Aperture is able to predict sites of viral integration and identify complex SVs involving novel insertions and repetitive sequences in real patient data. Aperture is freely available at https://github.com/liuhc8/Aperture. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B. To generate highcoverage, long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included .1,000 bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCCderived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations, and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV is a driver of genetic instability and provides a potential new mechanism for HCC development. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) is the causative driver of cervical cancer and a contributing risk factor of head and neck cancer and several anogenital cancers. HPV’s ability to induce genome instability contributes to its oncogenicity. HPV genes can induce genome instability in several ways, including modulating the cell cycle to favour proliferation, interacting with DNA damage repair pathways to bring high-fidelity repair pathways to viral episomes and away from the host genome, inducing DNA-damaging oxidative stress, and altering the length of telomeres. In addition, the presence of a chronic viral infection can lead to immune responses that also cause genome instability of the infected tissue. The HPV genome can become integrated into the host genome during HPV-induced tumorigenesis. Viral integration requires double-stranded breaks on the DNA; therefore, regions around the integration event are prone to structural alterations and themselves are targets of genome instability. In this review, we present the mechanisms by which HPV-dependent and -independent genome instability is initiated and maintained in HPV-driven cancers, both across the genome and at regions of HPV integration.

Human papillomavirus (HPV) is a sexually transmitted infection common among men and women of reproductive age worldwide. HPV viruses are associated with epithelial lesions and cancers. HPV infections have been shown to be significantly associated with many adverse effects in reproductive function. Infection with HPVs, specifically of high-oncogenic risk types (HR HPVs), affects different stages of human reproduction, resulting in a series of adverse outcomes: 1) reduction of male fertility (male infertility), characterized by qualitative and quantitative semen alterations; 2) impairment of couple fertility with increase of blastocyst apoptosis and reduction of endometrial implantation of trophoblastic cells; 3) defects of embryos and fetal development, with increase of spontaneous abortion and spontaneous preterm birth. The actual molecular mechanism(s) by which HPV infection is involved remain unclear. HPV-associated infertility as Janus, has two faces: one reflecting anti-HPV immunity, and the other, direct pathogenic effects of HPVs, specifically, of HR HPVs on the infected/HPV-replicating cells. Adverse effects observed for HR HPVs differ depending on the genotype of infecting virus, reflecting differential response of the host immune system as well as functional differences between HPVs and their individual proteins/antigens, including their ability to induce genetic instability/DNA damage. Review summarizes HPV involvement in all reproductive stages, evaluate the adverse role(s) played by HPVs, and identifies mechanisms of viral pathogenicity, common as well as specific for each stage of the reproduction process. [ABSTRACT FROM AUTHOR]

• DNA/chromosome- and extracellular matrix (ECM)-associated activities are key events during SV40T lentivirus-induced immortalization. • Many other transcription factors play more important roles than pRB in the SV40T-induced immortalization of MEFs, such as STAT1. • Viral integration may promote immortalization by disturbing the transcription of genes involved in DNA/chromosome- and ECM-associated activities. Lentivirus containing simian virus 40 large T antigen (SV40T) is routinely used to induce cell immortalization. However, the roles of viral integration itself in this progress is still controversial. Here, we transformed primary mouse embryonic fibroblasts (MEFs) with SV40T lentivirus and studied the roles of viral integration in the immortalization using RNA sequencing (RNA-seq) and whole genome sequencing (WGS). During the immortalization, differentially expressed genes (DGEs) are enriched in viral infection and several diverse activities. However, DEGs between immortalized and aging cells are significantly enriched in DNA/chromosome- and extracellular matrix (ECM)-associated activities. Gene regulatory network (GRN) analysis shows that although p53 is a key regulatory factor, many other transcription factors also play critical roles in the process, like STAT1. Of these DEGs, 32 genes have viral integration in their coding and/or regulatory regions. Our findings suggest that viral integration may promote SV40T-mediated immortalization by disturbing the expression of DNA/chromosome- and ECM-associated genes. [ABSTRACT FROM AUTHOR]

Abstract Background & Aims Although the prognosis of patients with occult hepatitis B virus (HBV) infection (OBI) is usually benign, a small portion may undergo cirrhosis and subsequently hepatocellular carcinoma (HCC). We studied the mechanism of life-long Integration of virus DNA into OBI host’s genome, of which may induce hepatocyte transformation. Methods We applied HBV capture sequencing on single cells from an OBI patient who, developed multiple HCC tumors and underwent liver resection in May 2013 at Tongji Hospital in China. Despite with the undetectable virus DNA in serum, we determined the pattern of viral integration in tumor cells and adjacent non-tumor cells and obtained the details of the viral arrangement in host genome, and furthermore the HBV integrated region in cancer genome. Results HBV captured sequencing of tissues and individual cells revealed that samples from multiple tumors shared two viral integration sites that could affect three host genes, including CSMD2 on chr1 and MED30/EXT1 on chr8. Whole genome sequencing further indicated one hybrid chromosome formed by HBV integrations between chr1 and chr8 that was shared by multiple tumors. Additional 50 poorly differentiated liver tumors and the paired adjacent non-tumors were evaluated and functional studies suggested up-regulated EXT1 expression promoted HCC growth. We further observed that the most somatic mutations within the tumor cell genome were common among the multiple tumors, suggesting that HBV associated, multifocal HCC is monoclonal in origin. Conclusion Through analyzing the HBV integration sites in multifocal HCC, our data suggested that the tumor cells were monoclonal in origin and formed in the absence of active viral replication, whereas the affected host genes may subsequently contribute to carcinogenesis.

Background: Several bioinformatics pipelines have been developed to detect sequences from viruses that integrate into the human genome because of the health relevance of these integrations, such as in the persistence of viral infection and/or in generating genotoxic effects, often progressing into cancer. Recent genomics and metagenomics analyses have shown that viruses also integrate into the genome of non-model organisms (i.e., arthropods, fish, plants, vertebrates). However, rarely studies of endogenous viral elements (EVEs) in non-model organisms have gone beyond their characterization from reference genome assemblies. In non-model organisms, we lack a thorough understanding of the widespread occurrence of EVEs and their biological relevance, apart from sporadic cases which nevertheless point to significant roles of EVEs in immunity and regulation of expression. The concomitance of repetitive DNA, duplications and/or assembly fragmentations in a genome sequence and intrasample variability in whole-genome sequencing (WGS) data could determine misalignments when mapping data to a genome assembly. This phenomenon hinders our ability to properly identify integration sites. Results: To fill this gap, we developed ViR, a pipeline which solves the dispersion of reads due to intrasample variability in sequencing data from both single and pooled DNA samples thus ameliorating the detection of integration sites. We tested ViR to work with both in silico and real sequencing data from a non-model organism, the arboviral vector Aedes albopictus. Potential viral integrations predicted by ViR were molecularly validated supporting the accuracy of ViR results. Conclusion: ViR will open new venues to explore the biology of EVEs, especially in non-model organisms. Importantly, while we generated ViR with the identification of EVEs in mind, its application can be extended to detect any lateral transfer event providing an ad-hoc sequence to interrogate. [ABSTRACT FROM AUTHOR]

Digestive tract cancers are frequent worldwide, despite efforts to control known predisposing factors, they keep increasing, this fact suggests that human papillomavirus (HPV) may play an important oncogenic role in these pathologies. In order to argue the incidence that HPV has in the genesis of gastrointestinal tract tumors and to determine if it is a possible causal agent, the scientific literature published to date on the subject was reviewed, and was found that 4.5% of all diagnosed cancers correspond to HPV; 12% are extra cervical, 20% esophagus, 31.9% colon adenomas, 43% colorectal adenocarcinoma, and 35% head and neck neoplasms (HNSCC). The types of HPV reported in the digestive tract are 6, 9, 11, 13, 16.18, 20, 24, 25, 30, 33, 51, 54, 57, DL 416, DL 428 and DL 436. Although, the studies show the HPV impact in the oncogenesis and its role as a prognostic marker in some of them, they do not have hard evidence that reveals this relationship, therefore demonstrating its integration would allow clarifying it. [ABSTRACT FROM AUTHOR]

A significant subset of carcinomas developed in the head and neck (H&NCs) are associated with specific human papillomaviruses (HPV) genotypes. In particular, 40–60% of oropharyngeal carcinoma cases are linked to HPV. Epidemiological studies have demonstrated that HPV oral infections are predominantly sexually transmitted and are more frequent among men (10–18%) than women (3.6–8.8%). Although there is a large diversity of HPV genotypes associated with H&NCs, HPV16 lineage represents 83% of the reported cases. The prognostic value of HPV as a biological parameter is well recognized. However, the use of HPV DNA as a diagnostic and/or predictive marker is not fully developed. Recent data reporting the physical state of the HPV genome in tumors have shown that HPV DNA integration into the tumor cell genome could lead to the alteration of cellular genes implicated in oncogenesis. Most importantly, HPV DNA corresponds to a tumor marker that can be detected in the blood of patients. Profile of the HPV DNA molecular patterns in tumor cells using New Genome Sequencing-based technologies, allows the identification of highly specific tumor markers valuable for the development of innovative diagnostic and therapeutic approaches. This review will summarize recent epidemiological data concerning HPV-associated H&NCs, the genomic characterization of these tumors, including the presence of HPV DNA in tumor cells, and will propose perspectives for developing improved care of patients with HPV-associated H&NCs, based on the use of viral sequences as personalized tumor markers and, over the longer term, as a therapeutic target.

Hepatitis B Virus (HBV) DNA integrations into the human genome are considered major causative factors to HBV-associated hepatocellular carcinoma development. In the present study, we investigated whether HBV preferentially integrates parts of its genome in specific genes and evaluated the contribution of the integrations in HCC development per gene. We applied dedicated in-house developed pipelines on all of the available HBV DNA integration data and performed a statistical analysis to identify genes that could be characterized as hotspots of integrations, along with the evaluation of their association with HBV-HCC. Our results suggest that 15 genes are recurrently affected by HBV integrations and they are significantly associated with HBV-HCC. Further studies that focus on HBV integrations disrupting these genes are mandatory in order to understand the role of HBV integrations in clonal advantage gain and oncogenesis promotion, as well as to determine whether inhibition of the HBV-disrupted genes can provide a therapy strategy for HBV-HCC.

BackgroundAntiretroviral therapy (ART)-mediated immune reconstitution fails to restore the capacity of the immune system to spontaneously control human immunodeficiency virus (HIV) replication.MethodsA total of 23 HIV type 1 (HIV-1)-infected, virologically suppressed subjects receiving ART (CD4(+) T-cell count, >450 cells/μL) were randomly assigned to have 180 μg/week (for arm A) or 90 μg/week (for arm B) of pegylated (Peg) interferon alfa-2a added to their current ART regimen. After 5 weeks, ART was interrupted, and Peg-interferon alfa-2a was continued for up to 12 weeks (the primary end point), with an option to continue to 24 weeks. End points included virologic failure (viral load, ≥ 400 copies/mL) and adverse events. Residual viral load and HIV-1 DNA integration were also assessed.ResultsAt week 12 of Peg-interferon alfa-2a monotherapy, viral suppression was observed in 9 of 20 subjects (45%), a significantly greater proportion than expected (arm A, P = .0088; arm B, P = .0010; combined arms, P < .0001). Over 24 weeks, both arms had lower proportions of subjects who had viral load, compared with the proportion of subjects in a historical control group (arm A, P = .0046; arm B, P = .0011). Subjects who had a sustained viral load of

A significant subset of carcinomas developed in the head and neck (H&NCs) are associated with specific human papillomaviruses (HPV) genotypes. In particular, 40–60% of oropharyngeal carcinoma cases are linked to HPV. Epidemiological studies have demonstrated that HPV oral infections are predominantly sexually transmitted and are more frequent among men (10–18%) than women (3.6–8.8%). Although there is a large diversity of HPV genotypes associated with H&NCs, HPV16 lineage represents 83% of the reported cases. The prognostic value of HPV as a biological parameter is well recognized. However, the use of HPV DNA as a diagnostic and/or predictive marker is not fully developed. Recent data reporting the physical state of the HPV genome in tumors have shown that HPV DNA integration into the tumor cell genome could lead to the alteration of cellular genes implicated in oncogenesis. Most importantly, HPV DNA corresponds to a tumor marker that can be detected in the blood of patients. Profile of the HPV DNA molecular patterns in tumor cells using New Genome Sequencing-based technologies, allows the identification of highly specific tumor markers valuable for the development of innovative diagnostic and therapeutic approaches. This review will summarize recent epidemiological data concerning HPV-associated H&NCs, the genomic characterization of these tumors, including the presence of HPV DNA in tumor cells, and will propose perspectives for developing improved care of patients with HPV-associated H&NCs, based on the use of viral sequences as personalized tumor markers and, over the longer term, as a therapeutic target. [ABSTRACT FROM AUTHOR]

Approximately 10–15% of worldwide human cancers are attributable to viral infection. When operating as carcinogenic elements, viruses may act with various mechanisms, but the most important is represented by viral integration into the host genome, causing chromosome instability, genomic mutations, and aberrations. In canine species, few reports have described an association between viral integration and canine cancers, but more comprehensive studies are needed. The advancement of next-generation sequencing and the cost reduction have resulted in a progressive increasing of sequencing data in veterinary oncology offering an opportunity to study virome in canine cancers. In this study, we have performed viral detection and integration analyses using VirusFinder2 software tool on available whole-genome and whole-exome sequencing data of different canine cancers. Several viral sequences were detected in lymphomas, hemangiosarcomas, melanomas, and osteosarcomas, but no reliable integration sites were identified. Even if with some limitations such as the depth and type of sequencing, a restricted number of available nonhuman genomes software, and a limited knowledge on endogenous retroviruses in the canine genome, results are compelling. However, further experiments are needed, and similarly to feline species, dedicated analysis tools for the identification of viral integration sites in canine cancers are required.

Abstract Background Transfer of genetic material from microbes or viruses into the host genome is known as horizontal gene transfer (HGT). The integration of viruses into the human genome is associated with multiple cancers, and these can now be detected using next-generation sequencing methods such as whole genome sequencing and RNA-sequencing. Results We designed a novel computational workflow, HGT-ID, to identify the integration of viruses into the human genome using the sequencing data. The HGT-ID workflow primarily follows a four-step procedure: i) pre-processing of unaligned reads, ii) virus detection using subtraction approach, iii) identification of virus integration site using discordant and soft-clipped reads and iv) HGT candidates prioritization through a scoring function. Annotation and visualization of the events, as well as primer design for experimental validation, are also provided in the final report. We evaluated the tool performance with the well-understood cervical cancer samples. The HGT-ID workflow accurately detected known human papillomavirus (HPV) integration sites with high sensitivity and specificity compared to previous HGT methods. We applied HGT-ID to The Cancer Genome Atlas (TCGA) whole-genome sequencing data (WGS) from liver tumor-normal pairs. Multiple hepatitis B virus (HBV) integration sites were identified in TCGA liver samples and confirmed by HGT-ID using the RNA-Seq data from the matched liver pairs. This shows the applicability of the method in both the data types and cross-validation of the HGT events in liver samples. We also processed 220 breast tumor WGS data through the workflow; however, there were no HGT events detected in those samples. Conclusions HGT-ID is a novel computational workflow to detect the integration of viruses in the human genome using the sequencing data. It is fast and accurate with functions such as prioritization, annotation, visualization and primer design for future validation of HGTs. The HGT-ID workflow is released under the MIT License and available at http://kalarikrlab.org/Software/HGT-ID.html.

Introduction. Human herpes virus type 6 (HHV 6) can cause serious infectious complications in immunodeficient patients. It is also capable of integrating into the genome of the infected cell. Due to this, there can be a misdiagnosis between viral integration and active infection during laboratory diagnostics. Thus, determination of HHV 6 infection using proper laboratory tools is relevant. Also the data on viral interference of HHV 6 and other herpes viruses are very poor especially for patients with hematological malignancies. The aim of the study was to identify laboratory markers of HHV 6 and the form of infection in patients with hematological malignancies. Materials and methods. 98 patients with hematological malignancies positive for HHV 6 DNA during the infectious complication were enrolled in the study. Viral load in leukocytes and plasma of peripheral blood, antiviral M and G immunoglobulins and peripheral blood leukocytes count were evaluated. Results. The majority of patients (66 out of 98, 67.3%) showed laboratory signs of latent HHV 6. Integrated HHV 6 was suspected in 2 patients due to high viral load (1.5x105 copies and 1.7x105 copies), but it was not confirmed subsequently. Additional testing of HCMV and EBV in patients with laboratory signs of active HHV 6 infection revealed the superiority of monoinfection over mixed infection (20 of 32, 62.5%). In cases of mixed infection, the most common co-infectant was HCMV observed in 9 out of 12 (75%) cases. Mild leukopenia accompanied HHV 6 active infection. Conclusion. Laboratory signs of latent HHV 6 tend to be prevalent in patients with hematological malignancies. In patients with laboratory markers of active HHV 6, the monoinfection demonstrated the superiority over mixed one. In cases of mixed infection, HCMV appeared to be the most commonly co-infectant. No cases of an integrated form of HHV 6 have been observed. The viral load of HHV 6 in leukocytes and blood plasma is almost 3 times lower in patients with a mixed infection than with a monoinfection. Active replication of HHV 6 was accompanied with mild leukopenia.

The avian α-herpesvirus known as Marek’s disease virus (MDV) linearly integrates its genomic DNA into host telomeres during infection. The resulting disease, Marek’s disease (MD), is characterized by virally-induced lymphomas with high mortality. The temporal dynamics of MDV-positive (MDV+) transformed cells and expansion of MD lymphomas remain targets for further understanding. It also remains to be determined whether specific host chromosomal sites of MDV telomere integration confer an advantage to MDV-transformed cells during tumorigenesis. We applied MDV-specific fluorescence in situ hybridization (MDV FISH) to investigate virus-host cytogenomic interactions within and among a total of 37 gonad lymphomas and neoplastic splenic samples in birds infected with virulent MDV. We also determined single-cell, chromosome-specific MDV integration profiles within and among transformed tissue samples, including multiple samples from the same bird. Most mitotically-dividing cells within neoplastic samples had the cytogenomic phenotype of ‘MDV telomere-integrated only’, and tissue-specific, temporal changes in phenotype frequencies were detected. Transformed cell populations composing gonad lymphomas exhibited significantly lower diversity, in terms of heterogeneity of MDV integration profiles, at the latest stages of tumorigenesis (>50 days post-infection (dpi)). We further report high interindividual and lower intraindividual variation in MDV integration profiles of lymphoma cells. There was no evidence of integration hotspots into a specific host chromosome(s). Collectively, our data suggests that very few transformed MDV+ T cell populations present earlier in MDV-induced lymphomas (32–50 dpi), survive, and expand to become the dominant clonal population in more advanced MD lymphomas (51–62 dpi) and establish metastatic lymphomas.

The hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC), partly driven by viral integration and specific oncogenic HBV variants. However, the biological significance of HBV genomes within lymphoid cells (i.e., peripheral blood mononuclear cells, PBMCs) is unclear. Here, we collected available plasma, PBMC, liver, and tumor from 52 chronic HBV (CHB) carriers: 32 with HCC, 19 without HCC, and one with dendritic cell sarcoma, DCS. Using highly sensitive sequencing techniques, next generation sequencing, and AluPCR, we demonstrate that viral genomes (i.e., HBV DNA, RNA, and cccDNA), oncogenic variants, and HBV-host integration are often found in all sample types collected from 52 patients (including lymphoid cells and a DCS tumor). Viral integration was recurrently identified (n = 90 such hits) in genes associated with oncogenic consequences in lymphoid and liver cells. Further, HBV genomes increased in PBMCs derived from 7 additional (treated or untreated) CHB carriers after extracellular mitogen stimulation. Our study shows novel HBV molecular data and replication not only liver, but also within 63.8% of lymphoid cells analysed (including a representative lymphoid cell malignancy), that was enhanced in ex vivo stimulated PBMC. [ABSTRACT FROM AUTHOR]

Background: Host genome integration of HBV sequence is considered to be significant in HBV antigen expression and the development of hepatocellular carcinoma (HCC). Method: We developed a probe-based capture strategy to enrich integrated HBV DNA for deep-sequencing analysis of integration sites in paired patient samples derived from tumor, liver tissue adjacent to tumor, saliva and plasma, as a platform for exploring the correlation, significance and utility of detecting integrations in these sample types. Results: Most significantly, alpha fetoprotein levels significantly correlated to the amounts of integrations detected in tumor. Viral-host chimeric DNA fragments were successfully detected at high sequencing coverage in plasma rather than saliva samples from HCC patients, and each fragment of this type was only seen once in plasma from chronic hepatitis B patients. Almost all plasma chimeric fragments were derived from integrations in tumor rather than in adjacent liver tissues. Over 50% of them may produce viral-host chimeric transcripts according to deep RNA sequencing in paired tissue samples. Particularly, in patients with low HBV DNA level (< 250 UI/ml), the seemingly normal HBsAg titers may be explained by larger amounts of integrations detected. Meanwhile, we developed a strategy to predict integrants by pairing breakpoints for each integration event. Among four resolved viral patterns, the majority of Pattern I events (81.2%) retained the complete opening reading frame for HBV surface proteins. Conclusion: We achieve the efficient enrichment of plasma cell-free chimeric DNA from integration site, and demonstrate that chimeric DNA profiling in plasma is a promising noninvasive approach to monitor HBV integration in liver cancer development and to determine the ability of integrated sequences to express viral proteins that can be targeted, e.g. by immunotherapies. [ABSTRACT FROM AUTHOR]

Many viruses are capable of integrating in the human genome, particularly viruses involved in tumorigenesis. Viral integrations can be considered genetic markers for discovering virus-caused cancers and inferring cancer cell development. Next-generation sequencing (NGS) technologies have been widely used to screen for viral integrations in cancer genomes, and a number of bioinformatics tools have been developed to detect viral integrations using NGS data. However, there has been no systematic comparison of the methods or software. In this study, we performed a comprehensive comparative analysis of the designs, performance, functionality and limitations among the existing methods and software for detecting viral integrations. We further compared the sensitivity, precision and runtime of integration detection of four representative tools. Our analyses showed that each of the existing software had its own merits; however, none of them were sufficient for parallel or accurate virome-wide detection. After carefully evaluating the limitations shared by the existing methods, we proposed strategies and directions for developing virome-wide integration detection. [ABSTRACT FROM AUTHOR]

Hepatitis B Virus (HBV) is an Old World virus with a high mutation rate, which puts its origins in Africa alongside the origins of Homo sapiens, and is a member of the Hepadnaviridae family that is characterized by a unique viral replication cycle. It targets human hepatocytes and can lead to chronic HBV infection either after acute infection via horizontal transmission usually during infancy or childhood or via maternal–fetal transmission. HBV has been found in ~85% of HBV-related Hepatocellular Carcinomas (HCC), and it can integrate the whole or part of its genome into the host genomic DNA. The molecular mechanisms involved in the HBV DNA integration is not yet clear; thus, multiple models have been described with respect to either the relaxed-circular DNA (rcDNA) or the double-stranded linear DNA (dslDNA) of HBV. Various genes have been found to be affected by HBV DNA integration, including cell-proliferation-related genes, oncogenes and long non-coding RNA genes (lincRNAs). The present review summarizes the advances in the research of HBV DNA integration, focusing on the evolutionary and molecular side of the integration events along with the arising clinical aspects in the light of WHO’s commitment to eliminate HBV and viral hepatitis by 2030.

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.

The incidence of oropharyngeal cancer (OPC) is increasing remarkably among all head and neck cancers, mainly due to its association with the human papillomavirus (HPV). Most HPVs are eliminated by the host’s immune system; however, because HPV has developed an effective immune evasion mechanism to complete its replication cycle, a small number of HPVs are not eliminated, leading to persistent infection. Moreover, during the oncogenic process, the extrachromosomal HPV genome often becomes integrated into the host genome. Integration involves the induction and high expression of E6 and E7, leading to cell cycle activation and increased genomic instability in the host. Therefore, integration is an important event in oncogenesis, although the associated mechanism remains unclear, especially in HPV-OPC. In this review, we summarize the current knowledge on HPV-mediated carcinogenesis, with special emphasis on immune evasion and integration mechanisms, which are crucial for oncogenesis.

Abstract Background The possibility of integrating viral vectors to become a persistent part of the host genome makes them a crucial element of clinical gene therapy. However, viral integration has associated risks, such as the unintentional activation of oncogenes that can result in cancer. Therefore, the analysis of integration sites of retroviral vectors is a crucial step in developing safer vectors for therapeutic use. Results Here we present VISMapper, a vector integration site analysis web server, to analyze next-generation sequencing data for retroviral vector integration sites. VISMapper can be found at: http://vismapper.babelomics.org . Conclusions Because it uses novel mapping algorithms VISMapper is remarkably faster than previous available programs. It also provides a useful graphical interface to analyze the integration sites found in the genomic context.

The link between viruses and cancer has intrigued scientists for decades. Certain viruses have been shown to be vital in the development of various cancers by integrating viral DNA into the host genome and activating viral oncogenes. These viruses include the Human Papillomavirus (HPV), Hepatitis B and C Viruses (HBV and HCV), Epstein-Barr Virus (EBV), and Human T-Cell Leukemia Virus (HTLV-1), which are all linked to the development of a myriad of human cancers. Third-generation sequencing technologies have revolutionized our ability to study viral integration events at unprecedented resolution in recent years. They offer long sequencing capabilities along with the ability to map viral integration sites, assess host gene expression, and track clonal evolution in cancer cells. Recently, researchers have been exploring the application of Oxford Nanopore Technologies (ONT) nanopore sequencing and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) sequencing in cancer research. As viral integration is crucial to the development of cancer via viruses, third-generation sequencing would provide a novel approach to studying the relationship interlinking viral oncogenes, viruses, and cancer. This review article explores the molecular mechanisms underlying viral oncogenesis, the role of viruses in cancer development, and the impact of third-generation sequencing on our understanding of viral integration into the human genome., Competing Interests: WD and SY hold equity in Yihui Bio, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Ye, Wang, Bu, Luo, Deng, Zhang and Yin.)

The classical lytic infection theory along with large T antigen-mediated oncogenesis cannot explain the BK polyomavirus (BKPyV)-associated tumor secondary to BKPyV-associated nephropathy (BKVAN), viremia/DNAemia, and viruria after renal transplantation. This study performed virome capture sequencing and pathological examination on regularly collected urine sediment and peripheral blood samples, and BKVAN and tumor biopsy tissues of 20 patients with BKPyV-associated diseases of different stages. In the early noncancerous stages, well-amplified integration sites were visualized by in situ polymerase chain reaction, simultaneously with BKPyV inclusion bodies and capsid protein expression. The integration intensity, the proportion of microhomology-mediated end-joining integration, and host PARP-1 and POLQ gene expression levels increased with disease progression. Furthermore, multiomics analysis was performed on BKPyV-associated urothelial carcinoma tissues, identifying tandem-like structures of BKPyV integration using long-read genome sequencing. The carcinogenicity of BKPyV integration was proven to disturb host gene expression and increase viral oncoprotein expression. Fallible DNA double-strand break repair pathways were significantly activated in the parenchyma of BKPyV-associated tumors. Olaparib showed an antitumor activity dose-response effect in the tumor organoids without BRCA1/2 genes mutation. In conclusion, the dynamic viral integration patterns actively participate in the progression of BKPyV-associated diseases and thus could be a potential target for disease monitoring and intervention., (Copyright © 2023 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

The Asian tiger mosquito Aedes albopictus is contributing to the (re)-emergence of Chikungunya virus (CHIKV). To gain insights into the molecular underpinning of viral persistence, which renders a mosquito a life-long vector, we coupled small RNA and whole genome sequencing approaches on carcasses and ovaries of mosquitoes sampled 14 days post CHIKV infection and investigated the profile of small RNAs and the presence of vDNA fragments. Since Aedes genomes harbor nonretroviral Endogenous Viral Elements (nrEVEs) which confers tolerance to cognate viral infections in ovaries, we also tested whether nrEVEs are formed after CHIKV infection. We show that while small interfering (si)RNAs are evenly distributed along the full viral genome, PIWI-interacting (pi)RNAs mostly arise from a ~1000 bp window, from which a unique vDNA fragment is identified. CHIKV infection does not result in the formation of new nrEVEs, but piRNAs derived from existing nrEVEs correlate with differential expression of an endogenous transcript. These results demonstrate that all three RNAi pathways contribute to the homeostasis during the late stage of CHIKV infection, but in different ways, ranging from directly targeting the viral sequence to regulating the expression of mosquito transcripts and expand the role of nrEVEs beyond immunity against cognate viruses.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. Almost half of HCC cases are associated with hepatitis B virus (HBV) infections, which often lead to HBV sequence integrations in the human genome. Accurate identification of HBV integration sites at a single nucleotide resolution is critical for developing a better understanding of the cancer genome landscape and of the disease itself. Here, we performed further analyses and characterization of HBV integrations identified by our recently reported VIcaller platform in recurrent or known HCC genes (such as TERT, MLL4, and CCNE1) as well as non-recurrent cancer-related genes (such as CSMD2, NKD2, and RHOU). Our pathway enrichment analysis revealed multiple pathways involving the alcohol dehydrogenase 4 gene, such as the metabolism pathways of retinol, tyrosine, and fatty acid. Further analysis of the HBV integration sites revealed distinct patterns involving the integration upper breakpoints, integrated genome lengths, and integration allele fractions between tumor and normal tissues. Our analysis also implies that the VIcaller method has diagnostic potential through discovering novel clonal integrations in cancer-related genes. In conclusion, although VIcaller is a hypothesis free virome-wide approach, it can still be applied to accurately identify genome-wide integration events of a specific candidate virus and their integration allele fractions.

Different studies have shown that HPV16‐positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome‐wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty‐three fresh‐frozen HPV‐16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo‐keto‐reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV‐positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non‐hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV‐positive (p ≤0.001) and ‐negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV‐negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment. What's new? Oropharyngeal squamous cell carcinoma (OPSCC) is often associated with high‐risk human papillomavirus infection, especially HPV16, but the clinical relevance of viral integration in a subset of these tumors remains unclear. Here the authors describe transcriptional signatures of metabolic reprogramming including increased expression of the aldo‐keto‐reductases AKR1C1 and AKR1C3 proteins in OPSCC cells regardless of HPV status. Tumors with upregulation of these proteins were strongly associated with poor prognosis suggesting prognostic as well as therapeutic implications for OPSCC patients beyond HPV16 infection. [ABSTRACT FROM AUTHOR]