Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.
- MeSH
- biosenzitivní techniky metody MeSH
- DNA vazebné proteiny genetika MeSH
- DNA virů genetika MeSH
- elektrochemické techniky * metody MeSH
- genom virový MeSH
- infekce papilomavirem * virologie MeSH
- integrace viru * genetika MeSH
- lidé MeSH
- lidský papilomavirus 16 * genetika MeSH
- messenger RNA * genetika MeSH
- nádory děložního čípku * virologie MeSH
- onkogenní proteiny virové * genetika MeSH
- Papillomavirus E7 - proteiny * genetika MeSH
- progrese nemoci MeSH
- RNA virová genetika MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Endogénne retrovírusy (ERV) sú genetické elementy, ktoré boli integrované do genómu hostiteľa pred viac ako 100 miliónmi rokov. Ich integrácia prebehla v zárodočných bunkách, čím sa v ľudskej populácii zabezpečil ich prenos z generácie na generáciu. V súčasnosti sa predpokladá, že tvoria až 8 % ľudského genómu. V priebehu evolúcie došlo v endogénnych retrovírusoch ku hromadeniu rôznych mutácii, čo viedlo k ich znefunkčneniu, a preto sa v minulosti považovali za odpadovú DNA. V posledných rokoch sa však ukazuje, že nie sú úplne nefunkčné. S pribúdajúcimi analýzami ľudského genómu sa odhaľujú ich potenciálne úlohy aj v ľudskom organizme.
Endogenous retroviruses (ERVs) are genetic elements that were integrated into the host genome more than 100 million years ago. Their integration took place in germ cells, ensuring their vertical transmission in the human population. They are currently thought to make up to 8 % of the human genome. During evolution, various mutations have accumulated in endogenous retroviruses, leading to their dysfunction, and were therefore considered as junk DNA in the past. However, in recent years it has turned out that they are not completely dysfunctional. With more data becoming available from human genome analyses, their potential roles in the human body are being revealed.
- MeSH
- autoimunitní nemoci etiologie virologie MeSH
- endogenní retroviry * genetika metabolismus MeSH
- genom lidský genetika MeSH
- genom virový genetika MeSH
- integrace viru genetika MeSH
- lidé MeSH
- Retroviridae - proteiny onkogenní genetika metabolismus MeSH
- Retroviridae genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Most retroviruses preferentially integrate into certain genomic locations and, as a result, their genome-wide integration patterns are non-random. We investigate the epigenetic landscape of integrated retroviral vectors and correlate it with the long-term stability of proviral transcription. Retroviral vectors derived from the avian sarcoma/leukosis virus expressing the GFP reporter were used to transduce the human myeloid lymphoblastoma cell line K562. Because of efficient silencing of avian retrovirus in mammalian cells, only ∼3% of established clones displayed stable proviral expression. We analyzed the vector integration sites in non-selected cells and in clones selected for the GFP expression. This selection led to overrepresentation of proviruses integrated in active transcription units, with particular accumulation in promoter-proximal areas. In parallel, we investigated the integration of vectors equipped with an anti-silencing CpG island core sequence. Such modification increased the frequency of stably expressing proviruses by one order. The modified vectors are also overrepresented in active transcription units, but stably expressed in distal parts of transcriptional units further away from promoters with marked accumulation in enhancers. These results suggest that integrated retroviruses subject to gradual epigenetic silencing during long-term cultivation. Among most genomic compartments, however, active promoters and enhancers protect the adjacent retroviruses from transcriptional silencing.
- MeSH
- Alpharetrovirus genetika MeSH
- buněčné linie MeSH
- buňky K562 MeSH
- CpG ostrůvky genetika MeSH
- epigeneze genetická MeSH
- genetická transkripce * MeSH
- genetické vektory genetika MeSH
- integrace viru genetika MeSH
- lidé MeSH
- promotorové oblasti (genetika) genetika MeSH
- proviry genetika MeSH
- umlčování genů MeSH
- zesilovače transkripce genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.
- MeSH
- infekce papilomavirem komplikace virologie MeSH
- integrace viru genetika MeSH
- lidé MeSH
- nádory hlavy a krku mortalita virologie MeSH
- prognóza MeSH
- proporcionální rizikové modely MeSH
- Southernův blotting MeSH
- spinocelulární karcinom virologie MeSH
- tonzilární nádory virologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Do komplexu šestého lidského herpesviru (Human herpesvirus – 6; HHV-6) jsou řazeny dva blízce příbuzné a v populaci běžně se vyskytující viry HHV-6 A a HHV-6 B. Primoinfekce HHV-6 probíhá většinou již v časném dětském věku, a to jak asymptomaticky, tak u HHV-6 B také ve formě šesté exantémové nemoci. Po primoinfekci zůstává HHV-6 v organismu přítomen v latentní formě, s možností virových reaktivací. Článek popisuje v odborné veřejnosti méně známý fenomén chromozomální integrace HHV-6 (Ci-HHV-6), který se v běžné populaci vyskytuje s frekvencí přibližně 1% a který je na rozdíl od běžně se šířící infekce dědičný a bez dosud známého patologického dopadu na nosiče této integrované HHV-6 DNA. I proto je znalost klinických souvislostí přítomnosti Ci-HHV-6 zásadní pro správně vedenou terapii v situaci vysoké pozitivity při molekulárně-biologické detekci HHV-6.
Two closely related and commonly found human herpesviruses HHV-6 A and HHV-6 B are classified into the sixth human herpes virus complex (HHV-6). Primary infection with HHV-6 often takes place in early childhood and it can be either asymptomatic or manifests itself as sixth disease (caused by HHV-6 B). HHV-6 remains present in a latent form in the body with the potential for virus reactivation. The article points out the phenomenon of chromosomal integration of HHV‑6 (Ci-HHV-6) which is found in about 1% of the population and, unlike the commonly spread HHV-6 infection, has become hereditary, with its pathological potential in Ci-HHV-6 DNA carriers remaining unknown. Therefore, the focus on clinical consequences of Ci-HHV-6 is of high relevance to the therapeutic strategy for patients with high HHV-6 positivity in molecular biological tests.
- MeSH
- chybná diagnóza prevence a kontrola MeSH
- diferenciální diagnóza MeSH
- infekce roseoloviry diagnóza genetika terapie MeSH
- integrace viru genetika MeSH
- lidé MeSH
- lidské chromozomy genetika virologie MeSH
- lidský herpesvirus 6 * genetika patogenita MeSH
- molekulární biologie MeSH
- polymerázová řetězová reakce využití MeSH
- Check Tag
- lidé MeSH
HIV integrase (IN) catalyzes the insertion of proviral DNA into the host cell chromosome. While IN has strict sequence requirements for the viral cDNA ends, the integration site preference has been shown to be very diverse. Here, we mapped the HIV IN strand transfer reaction requirements using various short oligonucleotides (ON) that mimic the target DNA. Most double stranded DNA dodecamers served as excellent IN targets with variable integration efficiency depending mostly on the ON sequences. The preferred integration was lost with any changes in the geometry of the DNA double helical structures. Various hairpin-loop-forming ONs also served as efficient integration targets. Similar integration preferences were also observed for ONs, in which the nucleotide hairpin loop was replaced with a flexible aliphatic linker. The integration biases with all target DNA structures tested were significantly influenced by changes in the resulting secondary ON structures.
- MeSH
- dimerizace MeSH
- DNA virů genetika MeSH
- HIV-1 genetika MeSH
- HIV-integrasa genetika izolace a purifikace MeSH
- integrace viru genetika MeSH
- katalýza MeSH
- konformace nukleové kyseliny MeSH
- molekulární sekvence - údaje MeSH
- oligonukleotidy genetika MeSH
- sekvence nukleotidů MeSH
- substrátová specifita genetika MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- práce podpořená grantem MeSH
Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.
- MeSH
- akutní lymfatická leukemie mortalita terapie virologie MeSH
- akutní myeloidní leukemie mortalita terapie virologie MeSH
- cytomegalovirové infekce mortalita MeSH
- Cytomegalovirus izolace a purifikace MeSH
- dítě MeSH
- DNA virů analýza MeSH
- dospělí MeSH
- fatální výsledek MeSH
- financování organizované MeSH
- infekce roseoloviry genetika virologie MeSH
- integrace viru genetika MeSH
- lidé MeSH
- lidské chromozomy virologie MeSH
- lidský herpesvirus 6 genetika MeSH
- plíce virologie MeSH
- pneumonie mortalita virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- transplantace hematopoetických kmenových buněk MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
Chromosomal integration of human herpesvirus 6 (HHV-6) is a novel situation found in a small percentage of individuals. While active HHV-6 infection is treatable using antivirals, the abnormally high level of HHV-6 DNA found in chromosomal integration of HHV-6 (CI-HHV-6) is not affected by such drugs. Stored DNA samples taken originally for detection of fusion genes and minimal residual disease from 339 pediatric patients treated for leukemia in the Czech Republic between the years 1995-2007 were tested retrospectively. Using real-time quantitative PCR technology, the quantity of HHV-6 DNA detected was normalized to 100,000 human genome equivalents as assessed by quantitation of the albumin gene. HHV-6 DNA was detected in 107 samples from 91 patients (26.8%). In the majority of samples (99) only a minute level of normalized viral copies (NVCs) (median 1.84 NVCs) was detected. A high viral load of approximately 100,000 NVCs was detected in 5 patients (1.5%; median 140,150 NVCs), in all of whom were confirmed subsequently CI-HHV-6 by a detection of HHV-6 DNA in hair follicles or in the nails. In all but one patient with HHV-6 variant B, variant A of the virus was detected. None of the patients with CI-HHV-6 had complications attributable to HHV-6 infection. The prevalence of CI-HHV-6 in childhood leukemia does not differ from that published for other patients or healthy populations. Where high levels of HHV-6 DNA are present, CI-HHV-6 should be confirmed as soon as possible so that potentially toxic but ineffective antiviral treatment can be stopped. (c) 2008 Wiley-Liss, Inc.
- MeSH
- chromozomy virologie MeSH
- dítě MeSH
- DNA virů analýza genetika MeSH
- financování organizované MeSH
- herpetické infekce diagnóza epidemiologie virologie MeSH
- integrace viru genetika MeSH
- kojenec MeSH
- krev virologie MeSH
- lidé MeSH
- lidský herpesvirus 6 genetika izolace a purifikace MeSH
- lymfoblastická leukemie-lymfom z prekurzorových T-buněk virologie MeSH
- mladiství MeSH
- myeloidní leukemie virologie MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- prevalence MeSH
- retrospektivní studie MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH
- MeSH
- DNA vazebné proteiny genetika MeSH
- financování organizované MeSH
- geny ras genetika MeSH
- integrace viru genetika MeSH
- koncové repetice MeSH
- kuřecí embryo MeSH
- lidé MeSH
- mapování chromozomů MeSH
- nádory ledvin genetika virologie MeSH
- nemoci drůbeže genetika MeSH
- onkogenní proteiny genetika MeSH
- onkogeny genetika MeSH
- polymerázová řetězová reakce MeSH
- proviry genetika MeSH
- ptačí proteiny genetika MeSH
- transkripční faktor Twist MeSH
- tumor supresorové geny MeSH
- virus ptačí myeloblastózy genetika MeSH
- Wilmsův nádor genetika virologie MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- lidé MeSH
- zvířata MeSH
