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1.
PLoS Pathog ; 20(5): e1012261, 2024 May.
Article de Anglais | MEDLINE | ID: mdl-38805555

RÉSUMÉ

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin.


Sujet(s)
Poulets , Maladie de Marek , Télomère , Intégration virale , Latence virale , Animaux , Poulets/virologie , Télomère/génétique , Télomère/virologie , Maladie de Marek/virologie , Maladie de Marek/immunologie , Maladie de Marek/prévention et contrôle , Vecteurs génétiques , Herpèsvirus de type 1 du dindon/génétique , Herpèsvirus de type 1 du dindon/immunologie , Vaccins contre la maladie de Marek/immunologie , Vaccins contre la maladie de Marek/génétique , Génome viral , Herpèsvirus aviaire de type 2/génétique , Herpèsvirus aviaire de type 2/immunologie , Séquences répétées d'acides nucléiques , Maladies de la volaille/virologie , Maladies de la volaille/immunologie , Maladies de la volaille/prévention et contrôle
2.
J Med Virol ; 96(6): e29606, 2024 Jun.
Article de Anglais | MEDLINE | ID: mdl-38818708

RÉSUMÉ

Hepatitis B virus (HBV) integration exists throughout the clinical course of chronic hepatitis B (CHB). This study investigated the effects of long-term antiviral therapy on the level and profiles of transcriptionally active HBV integration. Serial liver biopsies and paired blood samples were obtained from 16, 16, and 22 patients with CHB at baseline, 78, and 260 weeks of entecavir monotherapy or combined with pegylated interferon alfa, respectively. Serum HBV biomarkers were longitudinally assessed. RNA-seq and HIVID2 program was used to identify HBV-host chimeric RNAs transcribed from integrated DNA. The counts of HBV integration reads were positively related to both serum HBV DNA levels (r = 0.695, p = 0.004) and HBeAg titers (r = 0.724, p = 0.021) at baseline, but the positive correlation exited only to the serum HBsAg levels after 260 weeks of antiviral therapy (r = 0.662, p = 0.001). After 78 weeks of antiviral therapy, the levels of HBV integration expression decreased by 12.25 folds from baseline. The viral junction points were enriched at the S and HBx genes after the long-term antiviral therapy. HBs-FN1 became one of the main transcripts, with the mean proportion of HBs-FN1 in all integrated expression increased from 2.79% at baseline to 10.54% at Week 260 of antiviral treatment. Antiviral therapy may reduce but not eliminate the HBV integration events and integration expression. Certain integration events, such as HBs-FN1 can persist in long-term antiviral treatment.


Sujet(s)
Antiviraux , ADN viral , Virus de l'hépatite B , Hépatite B chronique , Foie , Intégration virale , Humains , Hépatite B chronique/traitement médicamenteux , Hépatite B chronique/virologie , Antiviraux/usage thérapeutique , Mâle , Virus de l'hépatite B/génétique , Virus de l'hépatite B/effets des médicaments et des substances chimiques , Adulte , Femelle , Foie/virologie , Adulte d'âge moyen , ADN viral/sang , ADN viral/génétique , Guanine/analogues et dérivés , Guanine/usage thérapeutique , Interféron alpha/usage thérapeutique , Antigènes e du virus de l'hépatite virale B/sang , Antigènes de surface du virus de l'hépatite B/sang , Études longitudinales
3.
Methods Mol Biol ; 2807: 141-151, 2024.
Article de Anglais | MEDLINE | ID: mdl-38743226

RÉSUMÉ

To integrate with host chromatin and establish a productive infection, HIV-1 must translocate the viral Ribonucleoprotein (RNP) complex through the nuclear pore complex (NPC). Current assay to measure HIV-1 nuclear import relies on a transient byproduct of HIV-1 integration failure called 2-LTR circles. However, 2-LTR circles require complete or near-complete reverse transcription and association with the non-homologous end joining (NHEJ) machinery in the nucleus, which can complicate interpretation of 2-LTR circle formation as a measure of nuclear import kinetics. Here, we describe an approach to measure nuclear import of infectious HIV-1 particles. This involves chemically induced dimerization of Nup62, a central FG containing nucleoporin. Using this technique, nuclear import of infectious particles can be monitored in both primary and cell culture models. In response to host factor depletion or restriction factors, changes in HIV-1 nuclear import can be effectively measured using the nuclear import kinetics (NIK) assay.


Sujet(s)
Transport nucléaire actif , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Complexe protéique du pore nucléaire , Pore nucléaire , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Humains , Pore nucléaire/métabolisme , Complexe protéique du pore nucléaire/métabolisme , Cinétique , Noyau de la cellule/métabolisme , Infections à VIH/virologie , Infections à VIH/métabolisme , Intégration virale
4.
Zhonghua Gan Zang Bing Za Zhi ; 32(4): 375-379, 2024 Apr 20.
Article de Chinois | MEDLINE | ID: mdl-38733195

RÉSUMÉ

Hepatitis B virus (HBV) DNA integration occurs during the reverse transcription process of HBV replication, which develops in the early stages of HBV infection and accompanies the entire disease course. The integration of HBV DNA is detrimental to the attainment of clinical cure goals and also raises the risk of developing liver cancer. Theoretically, nucleos(t)ide analogs can reduce the synthesis of new double-stranded linear DNA, but there is no clearance function for hepatocytes that have already integrated HBV. Therefore, patients with serum HBV DNA-negative conversions still have the risk of developing liver cancer. As an immunomodulatory drug, interferon can not only inhibit viral replication but also inhibit or even eliminate existing clonally amplified hepatocytes carrying integrated HBV DNA fragments. However, there are currently few studies on the effects of nucleos(t)ide analogues and interferon therapy on HBV DNA integration. Thus, large-scale clinical studies are urgently needed for further clarification.


Sujet(s)
Antiviraux , Virus de l'hépatite B , Hépatite B , Humains , Antiviraux/usage thérapeutique , Antiviraux/pharmacologie , ADN viral , Hépatite B/traitement médicamenteux , Hépatite B/virologie , Virus de l'hépatite B/effets des médicaments et des substances chimiques , Virus de l'hépatite B/génétique , Interférons/usage thérapeutique , Intégration virale , Réplication virale/effets des médicaments et des substances chimiques
5.
Viruses ; 16(5)2024 04 25.
Article de Anglais | MEDLINE | ID: mdl-38793552

RÉSUMÉ

The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.


Sujet(s)
VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Macrophages , Lymphocytes T , Facteurs de clivage et de polyadénylation de l'ARN messager , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Humains , Facteurs de clivage et de polyadénylation de l'ARN messager/métabolisme , Facteurs de clivage et de polyadénylation de l'ARN messager/génétique , Lymphocytes T/virologie , Lymphocytes T/métabolisme , Cellules HeLa , Macrophages/virologie , Macrophages/métabolisme , Intégration virale , Noyau de la cellule/métabolisme , Protéines de capside/métabolisme , Protéines de capside/génétique , Infections à VIH/virologie , Infections à VIH/métabolisme , Capside/métabolisme
6.
J Med Virol ; 96(5): e29674, 2024 May.
Article de Anglais | MEDLINE | ID: mdl-38757834

RÉSUMÉ

Human Papillomaviruses (HPV) are a diverse family of non-enveloped dsDNA viruses that infect the skin and mucosal epithelia. Persistent HPV infections can lead to cancer frequently involving integration of the virus into the host genome, leading to sustained oncogene expression and loss of capsid and genome maintenance proteins. Microhomology-mediated double-strand break repair, a DNA double-stranded breaks repair pathway present in many organisms, was initially thought to be a backup but it's now seen as vital, especially in homologous recombination-deficient contexts. Increasing evidence has identified microhomology (MH) near HPV integration junctions, suggesting MH-mediated repair pathways drive integration. In this comprehensive review, we present a detailed summary of both the mechanisms underlying MH-mediated repair and the evidence for its involvement in HPV integration in cancer. Lastly, we highlight the involvement of these processes in the integration of other DNA viruses and the broader implications on virus lifecycles and host innate immune response.


Sujet(s)
Carcinogenèse , Papillomaviridae , Infections à papillomavirus , Humains , Papillomaviridae/pathogénicité , Papillomaviridae/génétique , Papillomaviridae/physiologie , Infections à papillomavirus/virologie , Infections à papillomavirus/complications , Intégration virale , Réparation de l'ADN , Cassures double-brin de l'ADN , ADN viral/génétique
7.
BMC Bioinformatics ; 25(1): 177, 2024 May 04.
Article de Anglais | MEDLINE | ID: mdl-38704528

RÉSUMÉ

BACKGROUND: Hepatitis B virus (HBV) integrates into human chromosomes and can lead to genomic instability and hepatocarcinogenesis. Current tools for HBV integration site detection lack accuracy and stability. RESULTS: This study proposes a deep learning-based method, named ViroISDC, for detecting integration sites. ViroISDC generates corresponding grammar rules and encodes the characteristics of the language data to predict integration sites accurately. Compared with Lumpy, Pindel, Seeksv, and SurVirus, ViroISDC exhibits better overall performance and is less sensitive to sequencing depth and integration sequence length, displaying good reliability, stability, and generality. Further downstream analysis of integrated sites detected by ViroISDC reveals the integration patterns and features of HBV. It is observed that HBV integration exhibits specific chromosomal preferences and tends to integrate into cancerous tissue. Moreover, HBV integration frequency was higher in males than females, and high-frequency integration sites were more likely to be present on hepatocarcinogenesis- and anti-cancer-related genes, validating the reliability of the ViroISDC. CONCLUSIONS: ViroISDC pipeline exhibits superior precision, stability, and reliability across various datasets when compared to similar software. It is invaluable in exploring HBV infection in the human body, holding significant implications for the diagnosis, treatment, and prognosis assessment of HCC.


Sujet(s)
Virus de l'hépatite B , Intégration virale , Virus de l'hépatite B/génétique , Humains , Intégration virale/génétique , Logiciel , Apprentissage profond , Mâle , Femelle , Hépatite B/génétique , Hépatite B/virologie , Tumeurs du foie/génétique , Tumeurs du foie/virologie , Biologie informatique/méthodes
8.
Viruses ; 16(4)2024 04 13.
Article de Anglais | MEDLINE | ID: mdl-38675945

RÉSUMÉ

The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.


Sujet(s)
Congrès comme sujet , Retroviridae , Intégration virale , Humains , Intégration virale/effets des médicaments et des substances chimiques , Retroviridae/physiologie , Retroviridae/effets des médicaments et des substances chimiques , Retroviridae/génétique , Infections à VIH/traitement médicamenteux , Infections à VIH/virologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Histoire du 21ème siècle , Histoire du 20ème siècle
9.
mBio ; 15(5): e0072924, 2024 May 08.
Article de Anglais | MEDLINE | ID: mdl-38624210

RÉSUMÉ

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.


Sujet(s)
ADN viral , Protéines des oncogènes viraux , Infections à papillomavirus , Tumeurs du col de l'utérus , Intégration virale , Humains , Femelle , Tumeurs du col de l'utérus/virologie , Tumeurs du col de l'utérus/génétique , Intégration virale/génétique , Protéines des oncogènes viraux/génétique , Protéines des oncogènes viraux/métabolisme , Infections à papillomavirus/virologie , Infections à papillomavirus/génétique , ADN viral/génétique , Papillomavirus humain de type 16/génétique , Papillomavirus humain de type 18/génétique , Lignée cellulaire tumorale , Oncogènes/génétique , Polyadénylation
10.
J Mol Biol ; 436(10): 168557, 2024 May 15.
Article de Anglais | MEDLINE | ID: mdl-38582148

RÉSUMÉ

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.


Sujet(s)
Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Intégration virale , Humains , ADN viral/composition chimique , Intégrase du VIH/composition chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Modèles moléculaires , Nucléoprotéines/composition chimique , Multimérisation de protéines
11.
Poult Sci ; 103(6): 103722, 2024 Jun.
Article de Anglais | MEDLINE | ID: mdl-38626691

RÉSUMÉ

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.


Sujet(s)
Poulets , Herpèsvirus aviaire de type 2 , Maladie de Marek , Maladies de la volaille , Animaux , Maladie de Marek/virologie , Maladie de Marek/épidémiologie , Poulets/virologie , Égypte/épidémiologie , Maladies de la volaille/virologie , Maladies de la volaille/épidémiologie , Herpèsvirus aviaire de type 2/génétique , Herpèsvirus aviaire de type 2/isolement et purification , Séquences répétées terminales , Virus de la réticuloendothéliose/génétique , Virus de la réticuloendothéliose/isolement et purification , Intégration virale , Génome viral
12.
J Vet Med Sci ; 86(6): 653-655, 2024 Jun 01.
Article de Anglais | MEDLINE | ID: mdl-38631888

RÉSUMÉ

The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.


Sujet(s)
Lymphocytes B , ADN viral , Leucose bovine enzootique , Virus de la leucémie bovine , Provirus , Animaux , Virus de la leucémie bovine/génétique , Leucose bovine enzootique/virologie , Bovins , Provirus/génétique , ADN viral/génétique , Lymphocytes B/virologie , Intégration virale , Prolifération cellulaire
13.
J Viral Hepat ; 31 Suppl 1: 26-34, 2024 04.
Article de Anglais | MEDLINE | ID: mdl-38606944

RÉSUMÉ

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.


Sujet(s)
Carcinome hépatocellulaire , Tumeurs du foie , Souris , Animaux , Humains , Tumeurs du foie/thérapie , Tumeurs du foie/génétique , Carcinome hépatocellulaire/anatomopathologie , Vecteurs génétiques , Plasmides , Thérapie génétique , Dependovirus/génétique , Dependovirus/métabolisme , Intégration virale
14.
J Med Virol ; 96(4): e29614, 2024 Apr.
Article de Anglais | MEDLINE | ID: mdl-38647071

RÉSUMÉ

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.


Sujet(s)
Épigenèse génétique , Virus de l'hépatite B , Intégration virale , Humains , Virus de l'hépatite B/génétique , Virus de l'hépatite B/physiologie , Intégration virale/génétique , ADN viral/génétique , Transcription génétique , Lignée cellulaire , Méthylation de l'ADN , Lignée cellulaire tumorale , Histone/génétique , Histone/métabolisme , Multi-omique
15.
Biochemistry (Mosc) ; 89(3): 462-473, 2024 Mar.
Article de Anglais | MEDLINE | ID: mdl-38648766

RÉSUMÉ

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3'-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription.


Sujet(s)
Domaine catalytique , Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Transcription inverse , Intégration virale , Intégrase du VIH/métabolisme , Intégrase du VIH/composition chimique , Intégrase du VIH/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Humains
16.
Blood ; 143(23): 2373-2385, 2024 Jun 06.
Article de Anglais | MEDLINE | ID: mdl-38452208

RÉSUMÉ

ABSTRACT: Gene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e-4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.


Sujet(s)
Dependovirus , Facteur VIII , Thérapie génétique , Vecteurs génétiques , Hémophilie A , Foie , Animaux , Chiens , Dependovirus/génétique , Hémophilie A/génétique , Hémophilie A/thérapie , Vecteurs génétiques/génétique , Foie/métabolisme , Foie/anatomopathologie , Mâle , Thérapie génétique/méthodes , Femelle , Facteur VIII/génétique , Techniques de transfert de gènes , Intégration virale , Transgènes , Modèles animaux de maladie humaine
17.
Curr Opin HIV AIDS ; 19(3): 110-115, 2024 05 01.
Article de Anglais | MEDLINE | ID: mdl-38457193

RÉSUMÉ

PURPOSE OF REVIEW: Elite controllers (ECs) and Posttreatment controllers (PTCs) represent a small subset of individuals who are capable of maintaining drug-free control of HIV plasma viral loads despite the persistence of a replication-competent viral reservoir. This review aims to curate recent experimental studies evaluating viral reservoirs that distinguish EC/PTC and may contribute to their ability to maintain undetectable viral loads in the absence of antiretroviral therapy. RECENT FINDINGS: Recent studies on ECs have demonstrated that integration sites of intact proviruses in EC/PTC are markedly biased towards heterochromatin regions; in contrast, intact proviruses in accessible and permissive chromatin were profoundly underrepresented. Of note, no such biases were noted when CD4 + T cells from EC were infected directly ex vivo, suggesting that the viral reservoir profile in EC is not related to altered integration site preferences during acute infection, but instead represents the result of immune-mediated selection mechanisms that can eliminate proviruses in transcriptionally-active euchromatin regions while promoting preferential persistence of intact proviruses in nonpermissive genome regions. Proviral transcription in such "blocked and locked" regions may be restricted through epigenetic mechanisms, protecting them from immune-recognition but presumably limiting their ability to drive viral rebound. While the exact immune mechanisms driving this selection process remain undefined, recent single-cell analytic approaches support the hypothesis that HIV reservoir cells are subject to immune selection pressure by host factors. SUMMARY: A "blocked and locked" viral reservoir profile may constitute a structural virological correlate of a functional cure of HIV-1 infection. Further research into the immunological mechanism promoting HIV-1 reservoir selection and evolution in EC/PTC is warranted and could inform foreseeable cure strategies.


Sujet(s)
Infections à VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Humains , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Provirus/génétique , Réplication virale , Lymphocytes T CD4+ , Intégration virale , Charge virale , Latence virale
18.
Zhonghua Gan Zang Bing Za Zhi ; 32(2): 164-167, 2024 Feb 20.
Article de Chinois | MEDLINE | ID: mdl-38514268

RÉSUMÉ

Chronic hepatitis B virus (HBV) infection will greatly contribute to raising the occurrence probability of cirrhosis and hepatocellular carcinoma in patients. Although existing antiviral treatment regimens have a certain effect on delaying disease progression and improving prognosis, it is still not effective in attaining functional cures. Hepatitis B virus DNA integration may be one of the reasons for this phenomenon. Therefore, this paper reviews the possible mechanisms of HBV DNA integration in maintaining chronic inflammation of the liver, evading existing antiviral treatment methods, and inducing hepatocellular carcinoma so as to further deepen the understanding of the role of HBV DNA integration in the occurrence and development of chronic hepatitis B, providing ideas and references for formulating better treatment strategies.


Sujet(s)
Carcinome hépatocellulaire , Hépatite B chronique , Hépatite B , Tumeurs du foie , Humains , Hépatite B chronique/traitement médicamenteux , Carcinome hépatocellulaire/génétique , Virus de l'hépatite B/génétique , Tumeurs du foie/génétique , ADN viral , Antiviraux/usage thérapeutique , Hépatite B/traitement médicamenteux , Intégration virale
19.
Gut ; 73(7): 1169-1182, 2024 Jun 06.
Article de Anglais | MEDLINE | ID: mdl-38395437

RÉSUMÉ

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.


Sujet(s)
Carcinome hépatocellulaire , Virus de l'hépatite B , Tumeurs du foie , Intégration virale , Carcinome hépatocellulaire/virologie , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/anatomopathologie , Tumeurs du foie/génétique , Tumeurs du foie/virologie , Tumeurs du foie/anatomopathologie , Virus de l'hépatite B/génétique , Humains , Intégration virale/génétique , Animaux , Souris , Mâle , Adulte d'âge moyen , Femelle , Adulte , Séquençage du génome entier , Variations de nombre de copies de segment d'ADN , Sujet âgé
20.
Biochimie ; 222: 9-17, 2024 Jul.
Article de Anglais | MEDLINE | ID: mdl-38373651

RÉSUMÉ

The cellular SFPQ protein is involved in several stages of the HIV-1 life cycle, but the detailed mechanism of its involvement is not yet fully understood. Here, the role of SFPQ in the early stages of HIV-1 replication has been studied. It is found that changes in the intracellular level of SFPQ affect the integration of viral DNA, but not reverse transcription, and SFPQ is a positive factor of integration. A study of the SFPQ interaction with HIV-1 integrase (IN) has revealed two diRGGX1-4 motifs in the N-terminal region of SFPQ, which are involved in IN binding. Substitution of a single amino acid residue in any of these regions led to a decrease in binding efficiency, while mutations in both motifs almost completely disrupted the SFPQ interaction with IN. The effect of the SFPQ mutants with impaired ability to bind IN on viral replication has been analyzed. Unlike the wild-type protein, the SFPQ mutants did not affect viral integration. This confirms that SFPQ influences the integration stage through direct interaction with IN. Our results indicate that the SFPQ/IN complex can be considered as a potential therapeutic target for the development of new inhibitors of HIV replication.


Sujet(s)
Intégrase du VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Facteur d'épissage associé à PTB , Intégration virale , Réplication virale , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Humains , Intégrase du VIH/métabolisme , Intégrase du VIH/génétique , Facteur d'épissage associé à PTB/métabolisme , Facteur d'épissage associé à PTB/génétique , Liaison aux protéines , Mutation , Cellules HEK293
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