Episomal Form Research Articles

Mitosis of hepatitis B virus-infected cells in vitro results in uninfected daughter cells

Background & AimsThe chronicity of HBV (and resultant liver disease) is determined by intrahepatic persistence of the HBV covalently closed circular DNA (cccDNA), an episomal form that encodes all viral transcripts. Therefore, cccDNA is a key target for new treatments, with the ultimate therapeutic aim being its complete elimination. Although established cccDNA molecules are known to be stable in resting hepatocytes, we aimed to understand their fate in dividing cells using in vitro models.MethodsWe infected HepG2-NTCP and HepaRG-NTCP cells with HBV and induced mitosis by passaging cells. We measured cccDNA copy number (by precise PCR assays) and HBV-expressing cells (by immunofluorescence) with wild-type HBV. We used reporter viruses expressing luciferase or RFP to track number of HBV-expressing cells over time after mitosis induction using luciferase assays and live imaging, respectively.ResultsIn all cases, we observed dramatic reductions in cccDNA levels, HBV-positive cell numbers, and cccDNA-dependent protein expression after each round of cell mitosis. The rates of reduction were highly consistent with mathematical models of a complete cccDNA loss in (as opposed to dilution into) daughter cells.ConclusionsOur results are concordant with previous animal models of HBV infection and show that HBV persistence can be efficiently overcome by inducing cell mitosis. These results support therapeutic approaches that induce liver turnover (e.g. immune modulators) in addition to direct-acting antiviral therapies to achieve hepatitis B cure.Lay summaryChronic hepatitis B affects 300 million people (killing 884,000 per year) and is incurable. To cure it, we need to clear the HBV genome from the liver. In this study, we looked at how the virus behaves after a cell divides. We found that it completely clears the virus, making 2 new uninfected cells. Our work informs new approaches to develop cures for chronic hepatitis B infections.

Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial (NCT02576795), liver biopsy samples were collected 2.6–4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.

The 'Why and How' of Cervical Cancers and Genital HPV Infection.

Knowing about the virology of human papillomavirus (HPV) including its structure, functions and mechanism of infection, helps in understanding the disease process and morphology of precancerous lesions for cervical cancer. Two types of HPV, low- and high-risk type, adopt different mechanisms of infection which cannot be differentiated on morphological basis. In addition to HPV infection, many other factors such as genetic predisposition, hormonal factors, host immune response, and multiple sexual partners can modify the course and progression of the disease. The viral genome comprises early and late proteins. These early and late genes are expressed in particular course of time after initial infection. Various products of early genes (E1–E7) coordinate for completion of viral life cycle in maturing squamous epithelium by utilizing replication factors and DNA polymerase enzyme of the host cell nucleus. The late genes are mainly concerned with packaging of the viral particles and their release through mature squamous cells. The episomal form of infection seen in the low-risk group of viruses results in productive infection whereas the integrated form seen in high-risk group of viruses is the basis of disruption of host cell growth cycle by inactivating two important tumor suppressor genes p53 and Rb gene by products of E6 and E7 genes. Cervical precancerous lesions and cancer are the resultant effect of the accumulation of mutations. This article discusses the virology of HPV, pathogenesis of HPV infection, and various other factors modifying the disease course.

Association of Human Papillomavirus and Epstein-Barr Virus Infection with Tonsil Cancer in Northeastern Thailand.

Background:Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are associated with head and neck cancer, including tonsil cancer (TC) in the oropharyngeal area. Increasing incidence of HPV and EBV infection in different cancer tissues of oropharynx in both epithelial and lymphoid tissues, have been reported. However, little is known about association of these tumor viruses with TC in the Thai population. Here, we investigated the prevalence of HPV and EBV infection in different histology of TC and their association with TC from Thai patients. Methods:Eighty-three exfoliated tonsil cells from non-cancer controls (NCC) and 65 formalin-fixed paraffin-embedded TC tissues (TC) that were histologically classified as tonsillar squamous-cell carcinoma (TSCC) or diffuse large B-cell lymphoma (DLBCL) were studied. Prevalence of HPV and EBV infection was determined by real-time PCR. HPV genotyping was performed by reverse line blot hybridization and HPV genome status was investigated by multiplex qPCR. Localization of EBV infection was determined by EBER in situ hybridization. Results:Infection of HPV and EBV in TC cases was 16.9% and 30.8%, whereas in exfoliated tonsil cells was 1.2% and 66.3% respectively. HPV infection was significantly higher in TSCC (30.6%) than DLBCL samples (13.8%). HPV58 was commonly detected and presented as an integrated form in TSCC, whereas only episomal form was found in DLBCL. EBV infection was significantly higher in DLBCL (44.8%) than TSCC samples (19.4%), and detected in both lower than among exfoliated tonsil cell samples (66.3%). By EBER in situ hybridization in TSCC, EBV infection localized both in epithelial cells and infiltrating lymphocytes. The co-occurrence of HPV and EBV infection was 11.11% and 13.79% of TSCC and DLBCL, respectively, was associated with well-differentiated TSCC. Conclusion:HPV and EBV infection was significantly involved in a specific TC tissue, and associated with a good clinical outcome in TSCC.

Overexpression of m6A-factors METTL3, ALKBH5, and YTHDC1 alters HPV16 mRNA splicing

We report that overexpression of the m6A-demethylase alkB homolog 5 RNA demethylase (ALKBH5) promoted production of intron retention on the human papillomavirus type 16 (HPV16) E6 mRNAs thereby promoting E6 mRNA production. ALKBH5 also altered alternative splicing of the late L1 mRNA by an exon skipping mechanism. Knock-down of ALKBH5 had the opposite effect on splicing of these HPV16 mRNAs. Overexpression of the m6A-methylase methyltransferase-like protein 3 (METLL3) induced production of intron-containing HPV16 E1 mRNAs over spliced E2 mRNAs and altered HPV16 L1 mRNA splicing in a manner opposite to ALKBH5. Overexpression of the nuclear m6A-“reader” YTH domain-containing protein 1 (YTHDC1), enhanced retention of the E6-encoding intron and promoted E6 mRNA production. We also show that HPV16 mRNAs are bound to YTHDC1 in human cells and that YTHDC1 affected splicing of HPV16 E6/E7 mRNAs produced from the episomal form of the HPV16 genome. Finally, we show that HPV16 mRNAs are m6A-methylated in tonsillar cancer cells. In summary, HPV16 mRNAs are methylated in HPV16-infected tonsillar cancer cells and overexpression of m6A-“writer” METTL3, m6A-“eraser” ALKBH5 and the m6A-“reader” YTHDC1 affected HPV16 mRNA splicing, suggesting that m6A plays an important role in the HPV16 gene expression program, at least in cancer cells.

Piper DNA virus 1 and 2 are endogenous pararetroviruses integrated into chromosomes of black pepper (Piper nigrum L).

The online version contains supplementary material available at 10.1007/s13337-021-00752-w.

Non-Random Pattern of Integration for Epstein-Barr Virus with Preference for Gene-Poor Genomic Chromosomal Regions into the Genome of Burkitt Lymphoma Cell Lines

Background: Epstein-Barr virus (EBV) is an oncogenic virus found in about 95% of endemic Burkitt lymphoma (BL) cases. In latently infected cells, EBV DNA is mostly maintained in episomal form, but it can also be integrated into the host genome, or both forms can coexist in the infected cells. Methods: In this study, we mapped the chromosomal integration sites of EBV (EBV-IS) into the genome of 21 EBV+ BL cell lines (BL-CL) using metaphase fluorescence in situ hybridization (FISH). The data were used to investigate the EBV-IS distribution pattern in BL-CL, its relation to the genome instability, and to assess its association to common fragile sites and episomes. Results: We detected a total of 459 EBV-IS integrated into multiple genome localizations with a preference for gene-poor chromosomes. We did not observe any preferential affinity of EBV to integrate into common and rare fragile sites or enrichment of EBV-IS at the chromosomal breakpoints of the BL-CL analyzed here, as other DNA viruses do. Conclusions: We identified a non-random integration pattern into 13 cytobands, of which eight overlap with the EBV-IS in EBV-transformed lymphoblastoid cell lines and with a preference for gene- and CpGs-poor G-positive cytobands. Moreover, it has been demonstrated that the episomal form of EBV interacts in a non-random manner with gene-poor and AT-rich regions in EBV+ cell lines, which may explain the observed affinity for G-positive cytobands in the EBV integration process. Our results provide new insights into the patterns of EBV integration in BL-CL at the chromosomal level, revealing an unexpected connection between the episomal and integrated forms of EBV.

Participation of Multifunctional RNA in Replication, Recombination and Regulation of Endogenous Plant Pararetroviruses (EPRVs)

Pararetroviruses, taxon Caulimoviridae, are typical of retroelements with reverse transcriptase and share a common origin with retroviruses and LTR retrotransposons, presumably dating back 1.6 billion years and illustrating the transition from an RNA to a DNA world. After transcription of the viral genome in the host nucleus, viral DNA synthesis occurs in the cytoplasm on the generated terminally redundant RNA including inter- and intra-molecule recombination steps rather than relying on nuclear DNA replication. RNA recombination events between an ancestral genomic retroelement with exogenous RNA viruses were seminal in pararetrovirus evolution resulting in horizontal transmission and episomal replication. Instead of active integration, pararetroviruses use the host DNA repair machinery to prevail in genomes of angiosperms, gymnosperms and ferns. Pararetrovirus integration – leading to Endogenous ParaRetroViruses, EPRVs – by illegitimate recombination can happen if their sequences instead of homologous host genomic sequences on the sister chromatid (during mitosis) or homologous chromosome (during meiosis) are used as template. Multiple layers of RNA interference exist regulating episomal and chromosomal forms of the pararetrovirus. Pararetroviruses have evolved suppressors against this plant defense in the arms race during co-evolution which can result in deregulation of plant genes. Small RNAs serve as signaling molecules for Transcriptional and Post-Transcriptional Gene Silencing (TGS, PTGS) pathways. Different populations of small RNAs comprising 21–24 nt and 18–30 nt in length have been reported for Citrus, Fritillaria, Musa, Petunia, Solanum and Beta. Recombination and RNA interference are driving forces for evolution and regulation of EPRVs.

Identification of endogenous and episomal piper yellow mottle virus from the leaves and berries of black pepper (Piper nigrum)

Piper yellow mottle virus (PYMoV) is the most frequently occurring virus causing mottle disease in black pepper plants in Sukabumi, West Java. As a pararetrovirus, PYMoV occurs in the plant as endogenous (inactive) and episomal (active, infectious) forms. Research was carried out to detect and identify PYMoV from leaf and berry samples by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) followed by sequencing and nucleotide sequence analysis. Both endogenous and episomal forms of PYMoV were successfully detected from leaf and berry samples, although the endogenous form was more numerous than the episomal form. Results from black pepper berries proved the existence of both forms within single plants. It was further confirmed that endogenous and episomal PYMoV from the leaves and berries of the same plant had identical sequences, indicating the same isolates. This study is the first report of an endogenous and episomal form of PYMoV in a single plant.

Viruses in the Nucleus.

Viral infection is intrinsically linked to the capacity of the virus to generate progeny. Many DNA and some RNA viruses need to access the nuclear machinery and therefore transverse the nuclear envelope barrier through the nuclear pore complex. Viral genomes then become chromatinized either in their episomal form or upon integration into the host genome. Interactions with host DNA, transcription factors or nuclear bodies mediate their replication. Often interfering with nuclear functions, viruses use nuclear architecture to ensure persistent infections. Discovering these multiple modes of replication and persistence served in unraveling many important nuclear processes, such as nuclear trafficking, transcription, and splicing. Here, by using examples of DNA and RNA viral families, we portray the nucleus with the virus inside.

First Report of Black Currant Reversion Virus and Gooseberry Vein Banding Associated Virus in Currants in Bosnia and Herzegovina

Currants (Ribes spp.) are infected by at least 30 viruses (Koloniuk et al. 2018; Teifion Jones 2002), yet two are associated with the most important virus diseases of the crop: black currant reversion virus (BRV) for currant reversion and gooseberry vein banding associated virus (GVBaV) for the homonymous disease (Teifion Jones et al. 2001). BRV belongs to subgroup C of the genus Nepovirus of the family Comoviridae, whereas GVBaV is a member of genus Badnavirus, family Caulimoviridae (Petrzik et al. 2012). To this date, there is no information on the presence of either viruses in currant in Bosnia and Herzegovina (BiH). Surveys were carried out to assess the occurrence and diversity of the two viruses using high-throughput sequencing (HTS). Thirteen currant specimens (one red, one white, and 11 black) were collected in spring 2018 from commercial orchards in three localities (Travnik, Teslic, and Bugojno). Only three plants exhibited virus-like symptoms: the red currant (Ribes rubrum) from Travnik displayed leaf and flower malformation; the white currant and one black currant (R. nigrum) from Teslic showed yellow patterns and chlorosis on the upper canopy. RNA was extracted from seven samples (white, red, and black currants; both symptomatic and asymptomatic) using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Germany) and further depleted of ribosomal RNA using the KAPA RNA Hyper+RiboErase HMR kit. Libraries were generated and sequenced on the Illumina NextSeq 500/550 platform in separate reactions. Sequence analysis using Virfind (Ho and Tzanetakis 2014) revealed the presence of BRV in the red currant plant with typical disease symptoms and an asymptomatic black currant (47 and 28 contigs respectively, from 105 to 1,584 bp) and GVBaV in an asymptomatic plant (17 contigs from 350 to 1,903 bp). Nucleic acids were extracted from all 13 samples using the Poudel et al. (2013) protocol that facilitated detection of both DNA and RNA viruses. Reverse transcription PCR (RT-PCR) and PCR analyses were performed with two pairs of virus-specific primers for BRV (F, 5′-GTAATACGCTGGTGTCTC-3′/R, 5′-GAAAGGACATTTCAGACTC-3′ and F, 5′-ATTTCGAGCTGTATGGTCG-3′/R, 5′-CTCGGAAGCAGTAGACCT-3′ [Lemmetty et al. 2001]) and for GVBaV (F, 5′-ACATCAAAGGGAAGGACAAC-3′/R, 5′-TCTAAAAGCATCCACTACCAC-3′ and F, 5′-TCAGACAGGCTCTCAACAATAC-3′/R, 5′-TTCTAAAAGCATCCACTACCAC-3′) (Jones et al. 2001). These assays identified an additional GVBaV-positive sample. All amplicons were sequenced, and BLAST analysis of the BRV sequences (GenBank accession nos. MK501976 to MK501978) and GVBaV (GenBank accession nos. MK519228 to MK519230) revealed 92 to 98% and 96 to 99% nucleotide identity to GenBank accessions AF321572 and JQ388488, respectively. The positive samples were further assessed for the presence of the viruses using northern-blot hybridization with a digoxigenin-labeled probe that targeted the nontranslated region of BRV RNA2 and conserved motifs in the ribonuclease H domain of GVBaV. The results were in line with HTS and PCR detection and with the symptoms expressed in the three symptomatic plants. In addition, the episomal form of GVBaV was confirmed by positive results in RT-PCR using DNase-treated total nucleic acids and rolling circle amplification. To our knowledge, this is the first time that BRV and GVBaV were detected in currants in BiH. Given the lack of a currant certification program in the country, this information will be used in the development of best management practices for control of the two viruses and their associated diseases in both field and nursery operations.

Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 major viral RNAs, i.e. preC, pg, preS1/2, S and HBx RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. We set up an HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that interferon-β treatment leads to a strong reduction of preC and pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA. Especially under infection conditions, it has been difficult to study the different hepatitis B virus transcripts in depth. This study introduces a new method that can be used in any standard lab to discriminate all hepatitis B viral transcripts in cell culture and in the serum of patients.

Mechanistic Signatures of Human Papillomavirus Insertions in Anal Squamous Cell Carcinomas.

The role of human papillomavirus (HPV) in anal squamous cell carcinoma (ASCC) carcinogenesis has been clearly established, involving the expression of viral oncoproteins and optional viral DNA integration into the host genome. In this article, we describe the various mechanisms and sites of HPV DNA insertion and assess their prognostic and predictive value in a large series of patients with HPV-positive ASCC with long-term follow-up. We retrospectively analyzed 96 tumor samples from 93 HPV-positive ASCC patients using the Capture-HPV method followed by Next-Generation Sequencing, allowing determination of HPV genotype and identification of the mechanisms and sites of viral genome integration. We identified five different mechanistic signatures of HPV insertions. The distribution of HPV signatures differed from that previously described in HPV-positive cervical carcinoma (p < 0.001). In ASCC samples, the HPV genome more frequently remained in episomal form (45.2%). The most common signature of HPV insertion was MJ-SC (26.9%), i.e., HPV–chromosomal junctions scattered at different loci. Functionally, HPV integration signatures were not associated with survival or response to treatment, but were associated with viral load (p = 0.022) and PIK3CA mutation (p = 0.0069). High viral load was associated with longer survival in both univariate (p = 0.044) and multivariate (p = 0.011) analyses. Finally, HPV integration occurred on most human chromosomes, but intragenic integration into the NFIX gene was recurrently observed (n = 4/51 tumors). Overall, the distribution of mechanistic signatures of HPV insertions in ASCC was different from that observed in cervical carcinoma and was associated with viral load and PIK3CA mutation. We confirmed recurrent targeting of NFIX by HPV integration, suggesting a role for this gene in ASCC carcinogenesis.

Whole Exome Sequencing Analysis in Chronic Lymphoproliferative Disorder of NK Cells (CLPD-NK) Patients Fails to Detect Significant Viral Load

Background. Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK) is a rare disease characterized by chronic NK cell lymphocytosis, typically equipped with a restricted pattern of activating killer immunoglobulin like receptors. Several lines of evidence point to a putative role of viral agents in the pathogenetic mechanisms accounting for NK cell proliferation. A role of Epstein Barr Virus (EBV) has been claimed in the aggressive form of NK cell proliferation [i.e. Aggressive NK Cell Leukemia (Kawa Ha et al, JCI 1989)]. At variance, in the indolent CLPD-NK, EBV DNA was seldom detected by PCR (Loughran TP Jr et al, Blood 1993), whereas we provided serologic evidence for previously experienced viral infections in the majority of CLPD-NK patients (Zambello R et al, Leukemia 1995). However, the role of infectious agents in the pathogenesis of chronic antigen stimulation of NK cells in CLPD-NK patients is still elusive. Methods. Several studies showed that whole exome sequencing (WES), as well as RNA-seq, can identify pathogens, particularly viruses, in cancer clones enabling the definition of possible integration sites in the host genome or their presence as episomal forms. In the present study, we searched viral sequences in WES data of a cohort of 10 CLPD-NK patients. High-depth (average 51 M reads) WES data of highly purified (&gt;97% purity) leukemic NK clone and autologous granulocytes, as control, from CLPD-NK patients were analyzed for pathogen discovery. Three different methods for virus detection were used: i) Centrifuge (Kim et al, Genome Research 2016), a metagenomic tool performing a wide search against GenBank viral and bacterial sequences, which has been recently used to detect EBV load in ANKL and NK T cell Lymphoma (NKTCL) tumor clones (Dufva et al. Nat Comm 2018); ii) VirusSeq (Chen et al., Bioinformatics 2013), a software to identify viruses and eventually their integration sites, grounding on its custom and relatively wide viral sequence database; and iii) ViFi (Nguyen et al. Nucleic Acids Res, 2018), which can find viral integration in the human genome, with an analysis focused on a narrow set of viruses (EBV, HPV, HBV) with well-established oncogenic potential. Results. First, as positive control two datasets were used: RNA-seq data of an HBV positive sample with known site of integration (provided by VirusSeq) and WES data of three NKTCL tumor and control clones with high, moderate or low EBV load in the tumor samples previously detected by Dufva et al. (Dufva O et al, Nat Comm 2018), publicly available at NCBI SRA. HBV presence was detected in the positive sample by all the three methods and our reanalysis of NKTCL samples with Centrifuge confirmed published results, detecting EBV in all tumoral samples and in none of the matched control samples. Next, analysis of samples from the 10 CLPD-NK patients with the three methods did not identify a significant viral load neither in tumor nor in control specimens. Neither VirusSeq nor ViFi found viruses in CLPD-NK samples, whereas, according to Centrifuge analysis, the human disease virus with the highest load in most patients was Varicella Zoster (Human alphaherpesvirus 3) but with low read count and, most relevant, without specificity for the cancer clone. Conclusions. Our very thorough WES analysis did not identify a significant and meaningful viral load in CLPD-NK samples, neither in leukemic clones nor in control cell population. Although not ruling out that a viral infection might play a role as starting event for inciting NK cell proliferation, our data do not support a role of viral infections in maintaining the proliferation in CLPD-NK. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Integration rates of human papilloma virus genome in a molecular survey on cervical specimens among Iranian patients

The human papilloma virus (HPV) as a major causative agent of different cancers is under investigation globally. In this study, we aim to investigate HPV infection in different cytological and pathological stages by different molecular methods, and then the viral genome integration of HPV-16 and -18 is determined by a specific real-time PCR method. The study included women who underwent liquid-based cytology. HPV PCR was conducted by MY09/11 universal primers, HPV genotyping was performed by INNO-LiPA HPV genotyping assay, and the viral genome status was defined by two real-time PCR assays. The statistics were calculated by SPSS v.22 software. In 1668 women included in the study with mean age±std. deviation of 35.6±0.7, HPV was detected in 632 (38%) participants. Following genotyping analyses, 16 HPV types and 713 strains were detected. HPV-16 and HPV-18 from high-risk types and HPV-6 and HPV-11 from low-risk types were the dominant types. We found HPV-16 strains in mixed form (58.8%), and of the HPV-18 strains, the episomal form was prevalent (92.9%). The statistics revealed significant presence of HPV-6 and within normal limits cases; HPV-16 and atypical squamous cells of undetermined significance; HPV-33 as well as HPV-39 and low-grade squamous intraepithelial lesion; HPV-6 and atypical squamous cells of undetermined significance; and HPV-35 as well as HPV-56 and squamous cell carcinoma. Our study showed high prevalence of HPV in low-grade cervical lesions, although it is associated with higher grades. The HPV molecular testing extra to cytology is recommended. HPV-16 and HPV-18 have different programs in genome integration in infected cells.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

Structural Basis for Cooperative Binding of EBNA1 to the Epstein-Barr Virus Dyad Symmetry Minimal Origin of Replication.

Epstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised oriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to the oriP Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the oriP Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.IMPORTANCE Epstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element in oriP is the essential minimal replicator of oriP Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.

Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions.

Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determinants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it’s association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues harboring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetectable pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promotion/maintanence of tumorigenic phenotype.

Adeno-associated virus in the liver: natural history and consequences in tumour development

ObjectiveAdeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%–80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed...

Carcinogenesis Associated with Human Papillomavirus Infection. Mechanisms and Potential for Immunotherapy.

Human papillomavirus (HPV) infection is responsible for approximately 5% of all cancers and is associated with 30% of all pathogen-related cancers. Cervical cancer is the third most common cancer in women worldwide; about 70% of cervical cancer cases are caused by the high-risk HPVs (HR HPVs) of genotypes 16 and 18. HPV infection occurs mainly through sexual contact; however, viral transmission via horizontal and vertical pathways is also possible. After HPV infection of basal keratinocytes or ecto-endocervical transition zone cells, viral DNA persists in the episomal form. In most cases, infected cells are eliminated by the immune system. Occasionally, elimination fails, and HPV infection becomes chronic. Replication of HPVs in dividing epithelial cells is accompanied by increased expression of the E6 and E7 oncoproteins. These oncoproteins are responsible for genomic instability, disruption of the cell cycle, cell proliferation, immortalization, and malignant transformation of HPV-infected cells. Besides, E6 and E7 oncoproteins induce immunosuppression, preventing the detection of HPV-infected and transformed cells by the immune system. HPV integration into the genome of the host cell leads to the upregulation of E6 and E7 expression and contributes to HPV-associated malignization. Prophylactic HPV vaccines can prevent over 80% of HPV-associated anogenital cancers. The vaccine elicits immune response that prevents initial infection with a given HPV type but does not eliminate persistent virus once infection has occurred and does not prevent development of the HPV-associated neoplasias, which necessitates the development of therapeutic vaccines to treat chronic HPV infections and HPV-associated malignancies.