Constitutive activation of transcription factor AP-1 in cervical cancer and suppression of human papillomavirus (HPV) transcription and AP-1 activity in HeLa cells by curcumin

The transcription factor AP-1 plays a central role in the transcriptional regulation of specific types of high-risk human papillomaviruses (HPVs) such as HPV16 and HPV18, which are etiologically associated with the development of cancer of the uterine cervix in women. In our study, we investigated the AP-1 binding activity and the expression pattern of different members of the AP-1 transcription factor family (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) in different grades of cervical lesions starting from mild dysplasia to invasive cervical tumors, including normal control tissues, using specific antibodies raised against each of the AP-1 members. Results indicate that though AP-1 showed high binding activity and the majority of its members were highly expressed in tumor tissues, there is a distinct pattern of gradual increase of c-fos and a concomitant decrease of fra-1 expression that perfectly match the progression of cervical lesions. While c-fos is highly expressed in invasive cervical tumor, the expression of fra-1 becomes almost nil or absent, but the reverse is true in both controls and early precancerous lesions. These findings corroborate the results obtained in the cervical cancer cell line, HeLa. Interestingly, despite very low or absent AP-1 binding in normal as well as in premalignant lesions, AP-1 transcription and its binding activity was found to be very high in malignant tissues showing a preferential heterodimerization of c-fos with JunB instead of its canonical dimerization partner c-jun. Both in vivo and in vitro studies demonstrate that the overexpression of c-fos and downregulation of fra-1 expression as well as a change in the dimerization pattern of the AP-1 complex seem to play a crucial role during progression to malignancy. In a previous study, we demonstrated that a synthetic antioxidant, pyrrolidine dithiocarbamate (PDTC) can selectively downregulate HPV expression in human keratinocytes and cervical cancer cell lines. Since a redox regulatory pathway is involved in the expression of HPV that can be modulated by an antioxidant-induced reconstitution of the AP-1 transcription complex, we have used curcumin (diferuloylmethane), an active component of the perennial herb turmeric, which is a potent antioxidant and is well-known for its antiinflammatory and anticarcinogenic activity, to modulate the transcription of AP-1 and HPV. We demonstrate for the first time that curcumin can selectively downregulate HPV18 transcription as well as the AP-1 binding activity in HeLa cells. Most interestingly, curcumin can reverse the expression dynamics of c-fos and fra-1 in this tumorigenic cell line, mimicking the expression pattern observed in normal controls or precancerous lesions. Observation of curcumin-mediated complete downregulation of AP-1 binding activity and reversal of c-fos/fra-1 transcription to a normal state in tumorigenic HeLa cells represents a novel mechanism that can control transcription of pathogenic HPVs during keratinocyte differentiation and progression of cervical cancer. Our study thus provides a basis for developing a novel therapeutic approach to control pathogenic HPV infection by using potent antioxidative agents, such as curcumin

Use of whole genome deep sequencing to define emerging minority variants in virus envelope genes in herpesvirus treated with novel antimicrobial K21.

New antivirals are required to prevent rising antimicrobial resistance from replication inhibitors. The aim of this study was to analyse the range of emerging mutations in herpesvirus by whole genome deep sequencing. We tested human herpesvirus 6 treatment with novel antiviral K21, where evidence indicated distinct effects on virus envelope proteins. We treated BACmid cloned virus in order to analyse mechanisms and candidate targets for resistance. Illumina based next generation sequencing technology enabled analyses of mutations in 85 genes to depths of 10,000 per base detecting low prevalent minority variants (<1%). After four passages in tissue culture the untreated virus accumulated mutations in infected cells giving an emerging mixed population (45-73%) of non-synonymous SNPs in six genes including two envelope glycoproteins. Strikingly, treatment with K21 did not accumulate the passage mutations; instead a high frequency mutation was selected in envelope protein gQ2, part of the gH/gL complex essential for herpesvirus infection. This introduced a stop codon encoding a truncation mutation previously observed in increased virion production. There was reduced detection of the glycoprotein complex in infected cells. This supports a novel pathway for K21 targeting virion envelopes distinct from replication inhibition

Selective Suppression of NF-kBp65 in Hepatitis Virus-Infected Pregnant Women Manifesting Severe Liver Damage and High Mortality

Fulminant hepatitis in Asian pregnant women is generally caused by hepatitis E virus infection, and extremely high mortality is most common in them. Decreased cell-mediated immunity is considered a major cause of death in these cases, but what exactly influences decreased immunity and high mortality specifically during pregnancy is not known. We used electrophoretic mobility shift assays, immunoblotting, and immunohistochemical analysis to study the expression and DNA binding activity of NF-kB p50 and NF-kB p65 in pregnant fulminant hepatic failure (FHF) patients and compared them with their nonpregnant counterparts. In both PBMC and postmortem liver biopsy specimens the DNA-binding activity of NF-kB was very high in samples from pregnant FHF patients compared with those from nonpregnant women as well as pregnant women with acute viral hepatitis (AVH) without FHF. Further dissection of the NF-kB complex in supershift assays demonstrated complete absence of p65 in the NF-kB complex, which is formed by homodimerization of the p50 component in pregnant FHF patients. Western blotting and immunohistochemical analysis of the expression of p50 and p65 proteins both showed higher levels of p50 expression and a complete absence or a minimal expression of p65, indicating its nonparticipation in NF-kB-dependent transactivation in pregnant FHF patients. We suggest that the exclusion of p65 from the NF-kB transactivation complex seems to be a crucial step that may cause deregulated immunity and severe liver damage, leading to the death of the patient. Our findings provide a molecular basis, for developing novel therapeutic approaches

Possible chromosomal and germline integration of human herpesvirus 7

Publisher Copyright: © 2017 The Authors. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.Peer reviewe

Tissue specific signature of HHV-6 infection in ME/CFS

First exposure to various human herpesviruses (HHVs) including HHV-6, HCMV and EBV does not cause a life-threatening disease. In fact, most individuals are frequently unaware of their first exposure to such pathogens. These herpesviruses acquire lifelong latency in the human body where they show minimal genomic activity required for their survival. We hypothesized that it is not the latency itself but a timely, regionally restricted viral reactivation in a sub-set of host cells that plays a key role in disease development. HHV-6 (HHV-6A and HHV-6B) and HHV-7 are unique HHVs that acquire latency by integration of the viral genome into sub-telomeric region of human chromosomes. HHV-6 reactivation has been linked to Alzheimer’s Disease, Chronic Fatigue Syndrome, and many other diseases. However, lack of viral activity in commonly tested biological materials including blood or serum strongly suggests tissue specific localization of active HHV-6 genome. Here in this paper, we attempted to analyze active HHV-6 transcripts in postmortem tissue biopsies from a small cohort of ME/CFS patients and matched controls by fluorescence in situ hybridization using a probe against HHV-6 microRNA (miRNA), miR-aU14. Our results show abundant viral miRNA in various regions of the human brain and associated neuronal tissues including the spinal cord that is only detected in ME/CFS patients and not in controls. Our findings provide evidence of tissue-specific active HHV-6 and EBV infection in ME/CFS, which along with recent work demonstrating a possible relationship between herpesvirus infection and ME/CFS, provide grounds for renewed discussion on the role of herpesviruses in ME/CFS

Chlamydia trachomatis Infection Induces Replication of Latent HHV-6

Peer reviewe

Time-resolved single-cell RNA-seq using metabolic RNA labelling

Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a method for massively parallel analysis of newly transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in ~55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms

Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27

Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3' processing factor CPSF. It thereby induces the assembly of a dead-end 3' processing complex, blocking mRNA 3' cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3' processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer

Correcting 4sU induced quantification bias in nucleotide conversion RNA-seq data

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA emerging during library preparation and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package

Understanding, diagnosing, and treating Myalgic encephalomyelitis/chronic fatigue syndrome - State of the art: Report of the 2nd international meeting at the Charité fatigue center.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a devastating disease affecting millions of people worldwide. Due to the 2019 pandemic of coronavirus disease (COVID-19), we are facing a significant increase of ME/CFS prevalence. On May 11th to 12th, 2023, the second international ME/CFS conference of the Charité Fatigue Center was held in Berlin, Germany, focusing on pathomechanisms, diagnosis, and treatment. During the two-day conference, more than 100 researchers from various research fields met on-site and over 700 attendees participated online to discuss the state of the art and novel findings in this field. Key topics from the conference included: the role of the immune system, dysfunction of endothelial and autonomic nervous system, and viral reactivation. Furthermore, there were presentations on innovative diagnostic measures and assessments for this complex disease, cutting-edge treatment approaches, and clinical studies. Despite the increased public attention due to the COVID-19 pandemic, the subsequent rise of Long COVID-19 cases, and the rise of funding opportunities to unravel the pathomechanisms underlying ME/CFS, this severe disease remains highly underresearched. Future adequately funded research efforts are needed to further explore the disease etiology and to identify diagnostic markers and targeted therapies