Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA.

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic-latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30-p53-DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.

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.

Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

Dissecting Herpes Simplex Virus 1-Induced Host Shutoff at the RNA Level.

Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection.IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.

Donor-to-recipient transmission and reactivation in a kidney transplant recipient of an inherited chromosomally integrated HHV-6A: Evidence and outcomes.

Human herpesvirus (HHV)-6A can be inherited and chromosomally integrated (iciHHV-6A), and donor-to-recipient transmission has been reported in solid organ transplant. However, when HHV-6A reactivation happens after transplant, the source of HHV-6A is often not evident and its pathogenicity remains unclear. Here, we present an exhaustive case of donor-to-recipient transmission and reactivation of iciHHV-6A through kidney transplant. The absence of HHV-6A genome from the nails of the recipient excluded a recipient-related iciHHV-6A. Viral loads > 7 log10 copies/106 cells in donor blood samples and similarities of U38, U39, U69, and U100 viral genes between donor, recipient, and previously published iciHHV-6A strains are proof of donor-related transmission. Detection of noncoding HHV-6 snc-RNA14 using fluorescence in situ hybridization analysis and immunofluorescence staining of HHV-6A gp82/gp105 late proteins on kidney biopsies showed evidence of reactivation in the transplanted kidney. Because HHV-6A reactivation can be life threatening in immunocompromised patients, we provide several tools to help during the complete screening and diagnosis.

Evaluation of liver failure in a pediatric transplant recipient of a liver allograft with inherited chromosomally integrated HHV-6B.

BACKGROUND: Active infections of human herpesvirus 6B (HHV-6B) are frequent in immunocompromised recipients after transplantation. Nevertheless, they need to be distinguished from latent inherited chromosomally integrated genomes (iciHHV-6) present in about 1% of the population to avoid unnecessary administration of toxic antivirals. METHODS: A 5-year-old child presented with acute liver allograft rejection associated with HHV-6 DNA in plasma, which led to an unfavorable outcome. We investigated the possibility of HHV-6 infection derived from an iciHHV-6 present in the donor's liver using molecular and histopathology studies in various tissues, including quantification of HHV-6 DNA, genotyping, sequencing for antiviral resistance genes, relative quantification of viral transcripts, and detection of gB and gH viral proteins. RESULTS: The presence of iciHHV-6B was evidenced in the donor with signs of reactivation in the gallbladder and transplanted liver (detection of HHV-6B mRNA and late proteins). This localized expression could have played a role in liver rejection. Low viral loads in the recipient's plasma, with identical partial U39 sequences, were in favor of viral DNA released from the transplanted liver rather than a systemic infection. CONCLUSIONS: Determination of iciHHV-6 status before transplantation should be considered to guide clinical decisions, such as antiviral prophylaxis, viral load monitoring, and antiviral therapy.

Chlamydia trachomatis impairs host base excision repair by downregulating polymerase ß.

Chlamydia trachomatis infections have been associated with ovarian cancer by several epidemiological studies. Here, we show that C. trachomatis-infected primary human ovarian epithelial cells display elevated oxidative DNA damage. Base excision repair, an important cellular mechanism to repair oxidative DNA lesions, was impaired in infected primary ovarian and in several other types of cells. Polymerase ß was downregulated in infected cells associated with upregulation of microRNA-499a (miR-499a). Stabilising polymerase ß by inhibiting miR-499a significantly improved repair. Moreover, downregulation of tumour suppressor p53 also resulted in attenuated repair in these cells. Thus, our data show that downregulation of polymerase ß by direct inhibition through miR-499a and downregulation of p53 debilitate the host-cell base excision repair during C. trachomatis infection.

Cytomegalovirus infection and allograft rejection among pediatric heart transplant recipients in the era of valganciclovir prophylaxis.

CMV infection remains a significant cause of morbidity among pediatric HTx recipients We explored the implications of CMV infection on post-transplant outcomes among CMV risk-stratified pediatric HTx recipients receiving VGC prophylaxis. Children who underwent HTx between January 2010 and October 2016 were stratified according to CMV risk at time of transplant and evaluated for evidence of post-transplant CMV infection, rejection, CAV, and graft loss. Among 97 children, 41 (42%) were considered HR or IR risk for CMV infection and received VGC prophylaxis. CMV DNAemia was observed in 34% of children, including 71% HR, 40% IR, and 18% LR individuals. Median time to CMV DNAemia following VGC prophylaxis was 32D among HR vs 277D in IR subjects (P = .042). No difference in overall graft loss was noted among groups, but CMV HR children had decreased rejection-free survival (3.5 years) compared to IR (6 years, P = .015) and LR children (8 years, P = .0003). CMV was noted on EMB in 13% of children but was not associated with increased CAV, rejection or graft loss. High-risk CMV status was associated with decreased time to CMV infection despite VGC prophylaxis, compared to IR, and decreased rejection-free survival times compared to both IR and LR recipients. Detection of CMV on EMB was not associated with increased rejection, CAV or graft loss. Additional studies are needed to explore the impact of CMV infection on rejection-free survival in HTx recipients.

Chronic viral infections in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

BACKGROUND AND MAIN TEXT: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and controversial clinical condition without having established causative factors. Increasing numbers of cases during past decade have created awareness among patients as well as healthcare professionals. Chronic viral infection as a cause of ME/CFS has long been debated. However, lack of large studies involving well-designed patient groups and validated experimental set ups have hindered our knowledge about this disease. Moreover, recent developments regarding molecular mechanism of pathogenesis of various infectious agents cast doubts over validity of several of the past studies. CONCLUSIONS: This review aims to compile all the studies done so far to investigate various viral agents that could be associated with ME/CFS. Furthermore, we suggest strategies to better design future studies on the role of viral infections in ME/CFS.

Possible chromosomal and germline integration of human herpesvirus 7.

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.

Reactivation of chromosomally integrated human herpesvirus-6 by telomeric circle formation.

More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.

Increased circulating fibronectin, depletion of natural IgM and heightened EBV, HSV-1 reactivation in ME/CFS and long COVID.

Myalgic Encephalomyelitis/ Chronic Fatigue syndrome (ME/CFS) is a complex, debilitating, long-term illness without a diagnostic biomarker. ME/CFS patients share overlapping symptoms with long COVID patients, an observation which has strengthened the infectious origin hypothesis of ME/CFS. However, the exact sequence of events leading to disease development is largely unknown for both clinical conditions. Here we show antibody response to herpesvirus dUTPases, particularly to that of Epstein-Barr virus (EBV) and HSV-1, increased circulating fibronectin (FN1) levels in serum and depletion of natural IgM against fibronectin ((n)IgM-FN1) are common factors for both severe ME/CFS and long COVID. We provide evidence for herpesvirus dUTPases-mediated alterations in host cell cytoskeleton, mitochondrial dysfunction and OXPHOS. Our data show altered active immune complexes, immunoglobulin-mediated mitochondrial fragmentation as well as adaptive IgM production in ME/CFS patients. Our findings provide mechanistic insight into both ME/CFS and long COVID development. Finding of increased circulating FN1 and depletion of (n)IgM-FN1 as a biomarker for the severity of both ME/CFS and long COVID has an immediate implication in diagnostics and development of treatment modalities.

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.

The Hidden Enemy Within: Non-canonical Peptides in Virus-Induced Autoimmunity.

Viruses play a key role in explaining the pathogenesis of various autoimmune disorders, whose underlying principle is defined by the activation of autoreactive T-cells. In many cases, T-cells escape self-tolerance due to the failure in encountering certain MHC-I self-peptide complexes at substantial levels, whose peptides remain invisible from the immune system. Over the years, contribution of unstable defective ribosomal products (DRiPs) in immunosurveillance has gained prominence. A class of unstable products emerge from non-canonical translation and processing of unannotated mammalian and viral ORFs and their peptides are cryptic in nature. Indeed, high throughput sequencing and proteomics have revealed that a substantial portion of our genomes comprise of non-canonical ORFs, whose generation is significantly modulated during disease. Many of these ORFs comprise short ORFs (sORFs) and upstream ORFs (uORFs) that resemble DRiPs and may hence be preferentially presented. Here, we discuss how such products, normally "hidden" from the immune system, become abundant in viral infections activating autoimmune T-cells, by discussing their emerging role in infection and disease. Finally, we provide a perspective on how these mechanisms can explain several autoimmune disorders in the wake of the COVID-19 pandemic.

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.

Chronic persistent HHV-6B infection after sulfasalazine-induced DRESS with demonstration of HHV-6 encoded small noncoding RNAs (sncRNAs) in Crohn's-like colitis: Case report.

A sulfasalazine-induced DRESS (Drug Reactivation with Eosinophilia and Systemic Symptoms) was complicated by a Crohn's-like colitis. We demonstrated HHV-6 reactivation with presence of HHV-6 DNA and small noncoding RNA in colonic lesions. This observation confirms the major role of HHV-6 reactivation in DRESS manifestations and the importance of looking for HHV-6 reactivation in DRESS.

Human Herpesvirus 6 Infection in Pediatric Liver Transplantation: Single-Center Study of Incidence, Outcomes, and Management.

BACKGROUND: Distinctions between HHV-6 primary infection in seronegative patients and HHV-6 reactivation in seropositive patients remains largely undescribed in pediatric liver transplant (LT) recipients. METHODS: We implemented pretransplant serology testing of HHV-6 in a large pediatric hospital and retrospectively assessed the incidence, manifestations and outcomes of HHV-6 infections over a 3-year period. RESULTS: Among 101 pediatric LT recipients, 96 had pretransplant HHV-6 serologies; 34 (35.4%) were seronegative and 62 (64.6%) seropositive. Posttransplantation, 8/25 (32%) seronegative patients had HHV-6 DNAemia (primary infection) compared to 2/48 (4%) seropositive patients (p=0.002). Compared to seropositive patients, seronegative patients with HHV-6 DNAemia were younger, and had symptoms of fever and/or elevated aminotransferases in association with higher viral loads, in the first month post-transplant. More than 90% of seronegative patients and 77.8% of seropositive patients had HHV-6 detected by PCR in liver biopsy obtained for concerns of allograft rejection, but most had no detectable concomitant DNAemia. Active replication of virus in the liver was confirmed by in situ hybridization in select cases. While HHV-6 infection occurred among patients on prophylaxis doses of antivirals for CMV, HHV-6 DNAemia and presenting symptoms resolved on treatment doses. CONCLUSIONS: HHV-6 DNA-emia occurred more frequently in seronegative pediatric LT recipients, usually in the early posttransplant period, and was subsequently detected in allograft biopsies. HHV-6 cannot be ruled out as a cause of hepatitis in the absence of allograft tissue testing and specialized virological assays, as HHV-6 may disrupt local allograft immune homeostasis while evading traditional screening methods using blood or plasma. The assessment of pre-transplant HHV-6 serological status may be important for risk stratification and post-transplant management of pediatric LT recipients.

Joining European Scientific Forces to Face Pandemics.

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.

Revisiting the genomes of herpesviruses.

Combining integrative genomics and systems biology approaches has revealed new and conserved features in the genome of human herpesvirus 6.