[Research progress on the effect of hepatitis B virus DNA integration on antiviral therapy].

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.

Microhomology-mediated repair machinery and its relationship with HPV-mediated oncogenesis.

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.

ViroISDC: a method for calling integration sites of hepatitis B virus based on feature encoding.

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.

Monitoring HIV-1 Nuclear Import Kinetics Using a Chemically Induced Nuclear Pore Blockade Assay.

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.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

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.

HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro.

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.

Integration and the risk of liver cancer-Is there a real risk?

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.

Multi-omics panoramic analysis of HBV integration, transcriptional regulation, and epigenetic modifications in PLC/PRF/5 cell line.

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.

Role of I182, R187, and K188 Amino Acid Residues in the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration.

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.

Brief Histories of Retroviral Integration Research and Associated International Conferences.

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.

"Block and lock" viral integration sites in persons with drug-free control of HIV-1 infection.

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.

[Research progress on the role of hepatitis B virus DNA integration in chronic hepatitis B].

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.

Long-read sequencing reveals the structural complexity of genomic integration of HPV DNA in cervical cancer cell lines.

BACKGROUND: Cervical cancer (CC) causes more than 311,000 deaths annually worldwide. The integration of human papillomavirus (HPV) is a crucial genetic event that contributes to cervical carcinogenesis. Despite HPV DNA integration is known to disrupt the genomic architecture of both the host and viral genomes in CC, the complexity of this process remains largely unexplored. RESULTS: In this study, we conducted whole-genome sequencing (WGS) at 55-65X coverage utilizing the PacBio long-read sequencing platform in SiHa and HeLa cells, followed by comprehensive analyses of the sequence data to elucidate the complexity of HPV integration. Firstly, our results demonstrated that PacBio long-read sequencing effectively identifies HPV integration breakpoints with comparable accuracy to targeted-capture Next-generation sequencing (NGS) methods. Secondly, we constructed detailed models of complex integrated genome structures that included both the HPV genome and nearby regions of the human genome by utilizing PacBio long-read WGS. Thirdly, our sequencing results revealed the occurrence of a wide variety of genome-wide structural variations (SVs) in SiHa and HeLa cells. Additionally, our analysis further revealed a potential correlation between changes in gene expression levels and SVs on chromosome 13 in the genome of SiHa cells. CONCLUSIONS: Using PacBio long-read sequencing, we have successfully constructed complex models illustrating HPV integrated genome structures in SiHa and HeLa cells. This accomplishment serves as a compelling demonstration of the valuable capabilities of long-read sequencing in detecting and characterizing HPV genomic integration structures within human cells. Furthermore, these findings offer critical insights into the complex process of HPV16 and HPV18 integration and their potential contribution to the development of cervical cancer.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

Establishment of a novel human T-cell leukemia virus type 1 infection model using cell-free virus.

The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.

The immediate-early protein 1 of human herpesvirus 6B interacts with NBS1 and inhibits ATM signaling.

Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.

HIV-1 capsid and viral DNA integration.

IMPORTANCE: HIV-1 capsid protein (CA)-independently or by recruiting host factors-mediates several key steps of virus replication in the cytoplasm and nucleus of the target cell. Research in the recent years have established that CA is multifunctional and genetically fragile of all the HIV-1 proteins. Accordingly, CA has emerged as a validated and high priority therapeutic target, and the first CA-targeting antiviral drug was recently approved for treating multi-drug resistant HIV-1 infection. However, development of next generation CA inhibitors depends on a better understanding of CA's known roles, as well as probing of CA's novel roles, in HIV-1 replication. In this timely review, we present an updated overview of the current state of our understanding of CA's multifunctional role in HIV-1 replication-with a special emphasis on CA's newfound post-nuclear roles, highlight the pressing knowledge gaps, and discuss directions for future research.

The HIV-1 Capsid-Targeted Inhibitor GSK878 Alters Selection of Target Sites for HIV DNA Integration.

Decades of effort have yielded highly effective antiviral agents to treat HIV, but viral strains have evolved resistance to each inhibitor type, focusing attention on the importance of developing new inhibitor classes. A particularly promising new target is the HIV capsid, the function of which can be disrupted by highly potent inhibitors that persist long term in treated subjects. Studies with such inhibitors have contributed to an evolving picture of the role of capsid itself-the inhibitors, like certain capsid protein (CA) amino acid substitutions, can disrupt intracellular trafficking to alter the selection of target sites for HIV DNA integration in cellular chromosomes. In this study, we compare effects on HIV integration targeting for two potent inhibitors-a new molecule targeting CA, GSK878, and the previously studied lenacapavir (LEN, formerly known as GS-6207). We find that both inhibitors reduce integration in active transcription units and near epigenetic marks associated with active transcription. A careful study of integration near repeated sequences indicated frequencies were also altered for integration within multiple repeat classes. One notable finding was increased integration in centromeric satellite repeats in the presence of LEN and GSK878, which is of interest because proviruses integrated in centromeric repeats have been associated with transcriptional repression, inducibility, and latency. These data add to the picture that CA protein remains associated with preintegration complexes through the point in infection during which target sites for integration are selected, and specify new aspects of the consequences of disrupting this mechanism.

Viral integration promotes SV40T-induced immortalization by disturbing the expression of DNA/chromosome- and ECM-associated functional genes.

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

The analysis of HPV integration sites based on nanopore sequencing and the profiling changes along the course of photodynamic therapy.

OBJECTIVE: To detect the HPV genotype and integration sites in patients with high-risk HPV infection at different stages of photodynamic therapy using nanopore technology and to evaluate the treatment effect. METHODS: Four patients with HPV infection were selected and subjected to photodynamic therapy, and cervical exfoliated cell was sampled at before treatment, after three courses of treatment and six courses of treatment, their viral abundance and insertion sites were analyzed by nanopore technology, and pathological examinations were performed before and after treatment. In this study, we developed a novel assay that combined viral sequence enrichment and Nanopore sequencing for identification of HPV genotype and integration sites at once. The assay has obvious advantages over qPCR or NGS-based methods, as it has better sensitivity after viral sequences enrichment and can generate long-reads (kb to Mb) for better detection rate of structure variations, moreover, fast turn-around time for real-time viral sequencing and analysis. RESULTS: The pathological grade was reduced in all four patients after photodynamic therapy. Virus has been cleared in two cases after treatment, the virus amount reduced after treatment but not completely cleared in one case, and two type viruses were cleared and one type virus persisted after treatment in the last patient with multiple infection. Viral abundance and the number of integration sites were positively correlated. Gene enrichment analysis showed complete viral clearance in 1 patient and 3 patients required follow-up. CONCLUSION: Nanopore sequencing can effectively monitor the abundance of HPV viruses and integration sites to show the presence status of viruses, and combined with the results of gene enrichment analysis, the treatment effect can be dynamically assessed.