Integration Of Viral DNA Research Articles

Cyclophilin A facilitates HIV-1 integration.

Interaction between the HIV-1 capsid and host cellular factors is essential for infection. However, the molecular details and functional consequences of viral-host factor interactions during HIV-1 infection are not fully understood. Over 30 years ago, Cyclophilin A (CypA) was identified as the first host protein to bind to the HIV-1 capsid. Now it is established that CypA-capsid interaction promotes reverse transcription and nuclear entry of HIV-1. In addition, CypA blocks TRIM5α-mediated restriction of HIV-1. In this report, we show that CypA promotes the post-nuclear entry step of HIV-1 integration by binding to the viral capsid. Notably, we show that CypA stimulates the viral DNA integration activity of the HIV-1 preintegration complex. Collectively, our studies identify a novel role of CypA during the early steps of HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

A case of squamous cell carcinoma of the lung misdiagnosed as tuberculosis. Is HPV infected by oral sex the causative agent?

BackgroundThe integration of high-risk human papilloma virus (HPV) DNA into the human genome has been implicated in cervical carcinogenesis and head and neck squamous cell cancer. However, its role in lung squamous cell carcinoma is not well understood. In addition, tuberculosis (TB) and lung cancer(LC) share similar clinical symptoms and imaging features, increasing the risk of misdiagnosis.Case PresentationThe patient presented with a 16-month history of hemoptysis, chest pain, and occasional fatigue, without fever, chills, or history of mechanical damage or autoimmune diseases. Examination revealed normal vital signs and laboratory parameters, except for a positive interferon-gamma release assay indicating tuberculosis infection. Bronchoscopic examinations identified congestion and edema of the tracheal wall, along with a tiny lesion in the right wall of the trachea. She had been misdiagnosed with tuberculosis. However, the diagnosis of squamous cell carcinoma was eventually confirmed by endoscopic biopsy. The patient’s macrogenomic second-generation sequencing (mNGS) of the bronchoscopic biopsy specimen was positive for HPV-16.The patient’s sex partner tested positive for HPV-16 in penile scrapings, indicating HPV transmission through oral sex.ConclusionsThis case highlights the potential for HPV infection acquired through oral sex to lead to lung squamous cell carcinoma. It emphasizes the importance of considering HPV-associated malignancies in patients with respiratory symptoms who engage in oral sexual behaviors.

Regulation of expression of unintegrated and integrated HIV-1 DNA: keeping the wolves at bay

The unintegrated HIV-1 DNAs formed by reverse transcription in the early hours after infection are subject to profound transcriptional silencing. The repression of expression of foreign DNA, as an aspect of the innate immune system, serves to restrict the activity of many invading pathogens. Newly formed retroviral DNAs are rapidly loaded with histones upon entry into the nucleus, and the repression of their expression is mediated by an array of host proteins that introduce histone modifications characteristic of heterochromatin, including histone methylation and histone deacetylation. Knockout or knockdown of expression or inhibition of these host factors can relieve the silencing, allowing for viral gene expression even in settings where HIV-1 DNA integration is blocked. When viral DNA integration is allowed, forming the integrated provirus, the silencing in most cases is dramatically relieved, leading to high levels of expression and formation of progeny virus. In some settings and cell types, silencing of the integrated DNA is maintained, or re-established, such that the infected cells retain a silent copy of the viral DNA without production of progeny virus. The basis for the typical switch from silent DNA to actively expressed DNA upon integration is not yet fully clear. This review will summarize the current understanding of the regulation of expression of unintegrated HIV-1 DNAs and the nature of the chromatin that is formed on the viral DNA, and will especially focus on the host machinery that establishes repressive heterochromatin-like structures on the unintegrated DNA. The activation of expression that normally occurs upon integration, and the special circumstances when viral DNA expression is not activated, will also be discussed. These cases can result in the formation of populations of infected cells carrying silent proviruses, which persist for decades in infected individuals in spite of antiviral therapy. This pool of latently infected cells can be stochastically reactivated to give rise to spreading virus whenever antiviral drugs are withdrawn, and constitute the barrier to a true “cure” of AIDS. The hope is that a deeper understanding of the regulation of expression of viral DNAs will lead to new means to prevent or control viremia and disease.

Unraveling the role of hepatitis B virus DNA integration in B-cell lymphomagenesis.

Studies have shown that hepatitis B virus (HBV)-associated B-cell non-Hodgkin lymphoma (NHL) constitutes a unique subgroup with distinct clinical features. It still leaves open the question of whether the integration of HBV DNA into the B-cell genome is a causal mechanism in the development of lymphoma. Using the hybridisation capture-based next generation sequencing and RNA sequencing, we characterised the HBV integration pattern in 45 HBV-associated B-cell NHL tumour tissues. A total of 354 HBV integration sites were identified in 13 (28.9%) samples, indicating the relatively low integration frequency in B-cell NHLs. High plasma HBV DNA loads were not associated with the existence of HBV integration. The insertion sites distributed randomly across all the lymphoma genome without any preferential hotspot neither at the chromosomal level nor at the genetic level. Intriguingly, most HBV integrations were nonclonal in B-cell NHLs, implying that they did not confer a survival advantage. Analysis of the paired diagnosis-relapse samples showed the unstable status of HBV integrations during disease progression. Furthermore, transcriptomic analysis revealed the limited biological impact of HBV integration. Our study provides an unbiased HBV integration map in B-cell NHLs, revealing the insignificant role of HBV DNA integration in B-cell lymphomagenesis.

HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration.

HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis.

The level of expression of HPV16 early transcripts is not associated with the natural history of cervical lesions.

The natural history of cervical cancer is closely linked to that of high-risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high-grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

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.

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

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.

IMPULSIVE DIFFERENTIAL EQUATION MODEL IN HIV-1 INHIBITION: ADVANCES IN DUAL INHIBITORS OF HIV-1 RT AND IN FOR THE PREVENTION OF HIV-1 REPLICATION

Reverse transcriptase (RT) and integrase (IN) are two pivotal enzymes in HIV-1 replication. RT converts the single-stranded viral RNA genome into double-stranded DNA and IN catalyzes the integration of viral double-stranded DNA into host DNA. Currently, dual inhibitors of HIV-1 RT and IN have become a hotspot in new anti-HIV drug research and development. A dual inhibitor of HIV-1 RT/IN does the same thing as the two independent drugs would do. In this paper, we develop a mathematical model comprising a system of nonlinear differential equations describing HIV-1 RT/IN catalyzed biochemical reactions based on Michaelis–Menten enzyme kinetic reaction. In the formulated model we incorporate HIV-1 RT/IN dual inhibitor which simultaneously works as a non-nucleoside RT inhibitor and IN inhibitor. To examine the efficacy of HIV-1 RT/IN dual inhibitor in the treatment of HIV-1 infection, we have introduced a one-dimensional impulsive differential equation model and determined an effective dosing regimen for applying the inhibitor numerically. Furthermore, the exact closed form solution of the impulsive differential equation model is carried out by using the Lambert W function and the local stability of the periodic solution is also obtained analytically. The results obtained from analytical as well as numerical studies provide a basic idea to investigate the minimum dose with the highest efficacy for administering HIV-1 RT/IN dual inhibitors to prevent HIV-1 infection.

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.

The cellular SFPQ protein as a positive factor in the HIV-1 integration

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.

PTEN Mediates the Silencing of Unintegrated HIV-1 DNA.

The integration of viral DNA into a host genome is an important step in HIV-1 replication. However, due to the high failure rate of integration, the majority of viral DNA exists in an unintegrated state during HIV-1 infection. In contrast to the robust expression from integrated viral DNA, unintegrated HIV-1 DNA is very poorly transcribed in infected cells, but the molecular machinery responsible for the silencing of unintegrated HIV-1 DNA remains poorly characterized. In this study, we sought to characterize new host factors for the inhibition of expression from unintegrated HIV-1 DNA. A genome-wide CRISPR-Cas9 knockout screening revealed the essential role of phosphatase and tensin homolog (PTEN) in the silencing of unintegrated HIV-1 DNA. PTEN's phosphatase activity negatively regulates the PI3K-Akt pathway to inhibit the transcription from unintegrated HIV-1 DNA. The knockout (KO) of PTEN or inhibition of PTEN's phosphatase activity by point mutagenesis activates Akt by phosphorylation and enhances the transcription from unintegrated HIV-1 DNA. Inhibition of the PI3K-Akt pathway by Akt inhibitor in PTEN-KO cells restores the silencing of unintegrated HIV-1 DNA. Transcriptional factors (NF-κB, Sp1, and AP-1) are important for the activation of unintegrated HIV-1 DNA in PTEN-KO cells. Finally, the knockout of PTEN increases the levels of active epigenetic marks (H3ac and H3K4me3) and the recruitment of PolII on unintegrated HIV-1 DNA chromatin. Our experiments reveal that PTEN targets transcription factors (NF-κB, Sp1, and AP-1) by negatively regulating the PI3K-Akt pathway to promote the silencing of unintegrated HIV-1 DNA.

Development of Potential Inhibitors for Human T-lymphotropic virus type I Integrase enzyme: A molecular modeling approach.

Integration of viral DNA into the host cell genome, carried out by the HTLV-1 integrase enzyme, is a crucial step in the Human T-lymphotropic virus type I (HTLV-1) life cycle. Thus, HTLV-1 integrase is considered an attractive therapeutic target; however, no clinically effective inhibitors are available to treat HTLV-1 infection. Objective The main objective was to identify potential drug-like compounds capable of effectively inhibiting HTLV-1 integrase activity. In this study, a model of HTLV-1 integrase structure and three integrase inhibitors (dolutegravir, raltegravir, and elvitegravir as scaffolds) were used for designing new inhibitors. Designed molecules were used as templates for virtual screening to retrieve new inhibitors from PubChem, ZINC15, and ChEMBL databases. Drug-likeness and docked energy of the molecules were investigated using the SWISS-ADME portal and GOLD software. The stability and binding energy of the complexes were further investigated using molecular dynamic (MD) simulation. Four novel potential inhibitors were developed using a structure-based design protocol and three compounds from virtual screening. They formed hydrogen bonding interactions with critical residues Asp69, Asp12, Tyr96, Tyr143, Gln146, Ile13, and Glu105. In addition, π stacking, halogen, and hydrogen bond interactions were seen between compounds (especially halogenated benzyl moieties) and viral DNA similar to those seen in parent molecules. MD simulation confirmed higher stability of the receptor-ligand complex than the ligand-free enzyme. Combing structure-based design and virtual screening resulted in identifying three drug-like molecules (PubChem CID_138739497, _70381610, and _140084032) that are suggested as lead compounds for developing effective drugs targeting HTLV-1 integrase enzyme.

HIV-1 capsid and viral DNA integration.

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.

Methylation of the Selected TP53 Introns in Advanced-Stage Ovarian Carcinomas.

Objective: Advanced-stage ovarian cancer (OC) is among the most fatal female genital tract neoplasms worldwide. Although different genetic mechanisms have been shown to be involved in ovarian carcinogenesis, the role of TP53 introns methylation is still unresolved. We performed methylation analysis of introns 1, 3, and 4 of the TP53 to identify patterns in primary stage III OCs, corresponding metastases, and healthy tissues. Methods: The study involved samples of paraffin-embedded tissues obtained from 80 patients with stage III OCs, who underwent surgery at the Department of Gynecology and Gynecologic Oncology of the Military Institute of Medicine in Warsaw, Poland. Altogether, 40 serous-type G2/3 OCs and 40 endometrioid-type G2/3 OCs were included. From the same patient, metastatic and normal tissues were simultaneously analyzed. As a control group, 80 tissue samples were collected from patients after bariatric operations. Human ovarian cancer A2780 cell line was also investigated. Total genomic DNA was isolated from paraffin-embedded tissue blocks and the methylation analysis was performed by bisulfite DNA conversion, DNA amplification with specific primers, cloning, and DNA sequencing. Results: All of the samples of intron 1 of TP53 were un-methylated in OCs, metastatic tissues, and in healthy tissues from the same patient. Also, no methylation of TP53 intron 1 was detected in cells from the human A2780 ovarian cancer cell line and in all samples from control group. In all samples, introns 3 and 4 of the TP53 were methylated in primary tumors, metastatic tissue, and in healthy tissue from the same patient, in human A2780 ovarian cell line, and in DNA samples from healthy patients. None of the clinicopatholocal features was related to the TP53 introns methylation status. Conclusions: Our data on TP53 introns methylation sheds new light on the mechanism of p53 activity for a better understanding of cancer biology. The study suggests the existence of an additional regulation rule of TP53 activity that involves demethylation-methylation mechanisms. Methylation at introns 3 and 4 may also overall help in protecting TP53 against damage by viral restrictases or viral DNA integration.

Bioinformatics analysis of immune characteristics in tumors with alternative carcinogenesis pathways induced by human papillomaviruses

BackgroundHuman papillomaviruses (HPVs) induce a subset of head and neck squamous cell carcinomas (HNSCC) and anogenital cancers, particularly cervical cancer (CC). The major viral proteins that contribute to tumorigenesis are the E6 and E7 oncoproteins, whose expression is usually enhanced after the integration of viral DNA into the host genome. Recently, an alternative tumorigenesis pathway has been suggested in approximately half of HNSCC and CC cases associated with HPV infection. This pathway is characterized by extrachromosomal HPV persistence and increased expression of the viral E2, E4, and E5 genes. The E6, E7, E5, and E2 proteins have been shown to modify the expression of numerous cellular immune-related genes. The antitumor immune response is a critical factor in the prognosis of HPV-driven cancers, and its characterization may contribute to the prediction and personalization of the increasingly used cancer immunotherapy.MethodsWe analyzed the immune characteristics of HPV-dependent tumors and their association with carcinogenesis types. Transcriptomic HNSCC and CC datasets from The Cancer Genome Atlas were used for this analysis.ResultsClustering with immune-related genes resulted in two clusters of HPV16-positive squamous cell carcinomas in both tumor types: cluster 1 had higher activation of immune responses, including stimulation of the antigen processing and presentation pathway, which was associated with higher immune cell infiltration and better overall survival, and cluster 2 was characterized by keratinization. In CC, the distribution of tumor samples into clusters 1 and 2 did not depend on the level of E2/E5 expression, but in HNSCC, most E2/E5-high tumors were localized in cluster 1 and E2/E5-low tumors in cluster 2. Further analysis did not reveal any association between the E2/E5 levels and the expression of immune-related genes.ConclusionsOur results suggest that while the detection of immune responses associated with preserved expression of genes encoding components of antigen processing and presentation machinery in HPV-driven tumors may be markers of better prognosis and an important factor in therapy selection, the type of carcinogenesis does not seem to play a decisive role in the induction of antitumor immunity.

A versatile method to profile hepatitis B virus DNA integration.

HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome. Illumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR. RAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.

High-risk human papillomavirus: prevalence, association with clinical and morphological characteristics of invasive cervical cancer

Purpose of the study. Analysis of contemporary literature sources dedicated to the study of characteristics of high oncogenic risk human papillomavirus (HPV), associative links of the virus’s molecular-genetic parameters with the clinical and morphological characteristics of invasive cervical cancer (ICC).Materials and methods. A literature search was conducted in electronic databases including the National Center for Biotechnology Information Search database (PubMed/Medline), ResearchGate, Scopus, Web of Science, Cancer Tomorrow, Global cancer observatory, Cochrane Library, eLIBRARY, DisserCat. The search covered the period from 1990 to 2022 and utilized keywords in Russian and their English translations: cervical cancer, high oncogenic risk HPV, HPV status, HPV genotype, multiple infections, DNA integration, viral load. More than 120 full-text sources were studied, in which the analysis of possible correlations between the parameters of human papillomavirus infection and the clinical and morphological characteristics of cervical cancer was carried out. In review included data from 57 publications. Results. According to most authors, more than 88 % of patients with invasive ICC are HPV-infected. The etiological structure is dominated by HPV 16 (70–72 %), 18 (13–15 %), and 45 (5–7 %) types. Non-detection of HPV HR in cervical cancer is associated, including with a false negative result of the study.The literature analysis showed the presence of an association between HPV status and age, morphological form of the tumor, viral load, high oncogenic risk HPV genotype, presence of deep stromal invasion, and metastatic involvement of lymph nodes. With regard to such molecular genetic parameters of HPV HR as viral load and HPV DNA integration, associative relationship with prognostic important clinical indicators of cervical tumor – the stage of the disease, the loco-regional prevalence of the tumor process, the histological type of tumor – remains the subject of discussion.Conclusion. Many publications report a correlative link between HPV status and high oncogenic risk HPV genotype with such an important factor affecting ICC treatment outcomes as the morphological form of the tumor. Ambiguous conclusions on the presence of an association between a number of molecular-genetic parameters of HPV infection with the main prognostic factor – stage of the disease (due to the heterogeneity of samples, the use of different test systems, and the insufficiently complete account of the main parameters of HPV infection, especially data on viral DNA integration), make it advisable to conduct further research on a representative group of patients with homogeneous diagnostic protocols to determine the most complete spectrum of parameters of high oncogenic risk HPV. In addition, the conducted literature analysis showed the promise of searching for predictors of the effectiveness of specialized treatment of patients with ICC among such HPV infection parameters as HPV status, high oncogenic risk HPV genotype, and the significantly associated physical status of viral DNA.