Bhupesh Prusty

University of Wuerzburg | JMU · Institute for Virology and Immune Biology

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 labe...

Human cytomegalovirus (HCMV) reactivation poses a substantial risk to transplant patients. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A*03:01 and HLA-B*15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes...

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 he...

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 dev...

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 v...

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 labe...

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....

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 hundreds...

Single-cell RNA genomics technologies are revolutionizing biomedical science by profiling single cells with unprecedented resolution, providing fundamental insights into the role of different cellular states and intercellular heterogeneity in health and disease. The combination of single-cell RNA sequencing (scRNA-seq) with metabolic RNA labelling...

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 cell...

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...

In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes...

For decades, human cytomegalovirus (HCMV) was thought to express ≈200 viral proteins during lytic infection. In recent years, systems biology approaches uncovered hundreds of additional viral gene products and suggested thousands of viral sites of transcription initiation. Despite all available data, the molecular mechanisms of HCMV gene regulation...

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel c...

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, manifest...

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 leve...

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 lo...

Objectives- To evaluate HHV-6 and PVB19 infection using PCR and IFA in the myocardium of pediatric patients with dilated cardiomyopathy (DCM) and the impact of viral persistence in the cardiac allograft after heart transplantation (HT). Methods- Multiplex droplet digital PCR was used to analyze the prevalence of viral sequences in myocardial sample...

Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. The virion host shut-off ( vhs ) protein plays a key role in this process by efficiently cleaving both host and viral mRNAs in a translation-initiation-dependent manner. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in transc...

Background: Cytomegalovirus (CMV) infection remains a significant cause of morbidity among pediatric heart transplant (HTx) recipients. We explored the implications of CMV infection on post-transplant outcomes among CMV risk stratified pediatric HTx recipients receiving 6M valganciclovir (VGC) prophylaxis. Methods: Retrospective review of pediatri...

HHV‐6A can be inherited and chromosomally integrated (iciHHV‐6A), and donor to recipient transmission has been reported in solid‐organ transplantation. However, when HHV‐6A reactivation happens after transplantation, the source of HHV‐6A is often not evident and its pathogenicity remains unclear. Here, we present an exhaustive case of donor‐to‐reci...

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including al...

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifactorial disorder with many possible triggers. Human herpesvirus (HHV)-6 and HHV-7 are two infectious triggers for which evidence has been growing. To understand possible causative role of HHV-6 in ME/CFS, metabolic and antiviral phenotypes of U2-OS cells were studied with and w...

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

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 th...

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuol...

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...

Background/Objectives: With mucocutaneous candidiasis being highly prevalent in HIV patients, the emergence of fluconazole-resistant Candida species forms a major challenge in treating and eradicating these infections. The objective of this study was to establish the antifungal activity of K21, a membrane-rupturing antimicrobial compound derived fr...

Objectives: To study the relation between asymptomatic CMV infection in the first year after transplant and subsequent rejection, CAV and graft loss in pediatric HTx recipients in the era of valganciclovir prophylaxis. Methods: Retrospective study was done of 97 first-time pediatric HTx recipients between 1.1.2010 and 10.30.2016. Asymptomatic CMV i...

Objectives- To evaluate HHV-6 and PVB19 infection using PCR and IFA in the myocardium of pediatric patients with dilated cardiomyopathy (DCM) and the prognostic implications of viral presence. Methods- Multiplex droplet digital PCR was used to analyze the prevalence of viral sequences in myocardial samples from 48 pediatric DCM patients and 10 con...

Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral t...

Purpose To study the relation between the presence and absence of asymptomatic CMV infection in the first year after heart transplant (HT) and subsequent rejection, cardiac allograft vasculopathy (CAV) and graft loss in the era of valganciclovir prophylactic treatment. Methods Retrospective study of 97 pediatric HT recipients between 2010 and 2016...

Purpose To evaluate if the virus found in the myocardium of dilated cardiomyopathy (DCM) pediatric patients persists in the cardiac allograft after heart transplant (HT), and its prognostic implications. Methods Multiplex polymerase chain reaction (PCR) was used to analyze myocardial samples from 48 DCM patients. Subsequently consecutive endomyoca...

Virology: a biomarker for HHV-6 reactivation The human herpesvirus 6 (HHV-6) expresses high levels of small non-coding RNA (sncRNA) molecules early in its reactivation from latency. Bhupesh Prusty from the University of Würzburg, Germany, and colleagues developed a laboratory system for studying HHV-6 infections in a human bone cancer cell line. T...

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...

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...

Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed...

HHV-6A and HHV-6B detection through the use of immunofluorescence staining was validated using FFPE sections of HHV-6A and HHV-6B-infected HSB-2 and Molt-3 cells respectively. Uninfected cells served as control.

Representative confocal microscopy images showing Purkinje cell-specific staining for HHV-6A (left) and HHV-6B (right). Cryo-sectioned cerebellar cortex samples were stained using antibodies raised against HHV-6A gp82/105 or HHV-6B OHV3 (Red). HHV-6 positive Purkinje cells are marked with white arrowhead. Purkinje cell specificity was tested using...

Immuno-fluorescence analysis to study HHV-6 infection in astrocytes and microglia within cerebellar cortex samples. (A) Representative images showing positive staining for astrocytes in HHV-6A and HHV-6B negative samples. (B) Representative images showing HHV-6A and -6B positivity only in Purkinje cells. Astrocytes were detected as negative for HHV...

Representative images from confocal image analysis to study HHV-6 infection in astrocytes within cerebellar cortex samples. Red arrowheads point to HHV-6 positive cells whereas white arrowheads point to astrocytes showing HHV-6 positive co-staining. The scale bars represent 200 μm.

Table showing the cause of death for both the cohorts 1 and 2. BPD, bipolar disorder; MDD, major depressive disorder; SCZ, schizophrenia; CON, controls.

Enriched pathway network for the toll-like receptors component of the GSEA results. A connection between nodes arises when there is sufficient similarity between the gene sets of these pathways (based on the Dice coefficient). The node colour represents the q-value (more intensive shades of red correspond to more significant q-values), whereas the...

Table showing median age of all the four types of cases from both cohorts 1 and 2. BPD, bipolar disorder; MDD, major depressive disorder; SCZ, schizophrenia; CON, controls.

Mean (+SD) virus load (per 10e6 cells) for total HHV-6, HHV-6A, and HHV-6B DNA for cohorts 1 and 2. Viral copy numbers were set as 0 for all samples with viral DNA copies below the limit of detection (LOD).

Summary of results from various types of tests carried out on both the cohort of samples.

Summary of gene set enrichment analysis (GSEA) analysis showing significantly enriched pathways.

Real-time based qPCR analysis showing quantification of HHV-6 genome equivalents per cell all the samples from both the cohorts 1 and 2. Mean Cq values as obtained from qPCR analysis and the subsequent calculations are described in detail.

Antibodies used for Immunohistochemistry and Western Blotting.

Chlamydia trachomatis, an obligate intracellular human pathogen, is a major cause of sexually transmitted diseases. Infections often occur without symptoms, a feature that has been attributed to the ability of the pathogen to evade the host immune response. We show here that C. trachomatis paralyses the host immune system by preventing the activati...

PI15 regulation and its effect on chlamydial growth.(A) PI15 is differentially regulated at transcriptional level during Chlamydia and /or HHV-6A infection. HeLa cells were infected with either C. trachomatis or HHV-6A or both for 24 h. Total RNA was extracted and was used for quantitative reverse transcriptase PCR to analyze PI15 mRNA level. hpi,...

Comparison of intra-cellular CPAF localization by different fixation methods. (A) Schematic diagram showing different planes of sectioning of images used for the study of PI15-CPAF co-localization. (B) Confocal images from CPAF or cHtrA stained Chlamydia infected cells fixed with PFA either at room temperature (RT) or at 37°C. DAPI staining was use...

PI15 localizes within the chlamydial inclusion lumen. (A) Validation of PI15-specific antibody staining by RNAi. HeLa cells were either transfected with siRNAs against PI15 or control siRNAs. 72 h post transfection, cells were infected with C. trachomatis. Cells were immunostained using PI15 or Chlamydia-specific Hsp60 (cHsp60) antibodies. Draq5 wa...

CQuantitative analysis of PI15 and CPAF co-localization within inclusions of persistent Chlamydia. (A) Confocal images showing CPAF and PI15 co-localization within two representative persistent chlamydial inclusions infected with wild type Chlamydia (WT Ctr) and HHV-6A. (B) Structured illumination micrograph of a Chlamydia infected HeLa cell immuno...

Functional interaction of CPAF and PI15. (A) CPAF zymogen co-immunoprecipitates with PI15. Co-IP experiments were carried out using T-REx-293 cells that inducibly express PI15-Flag. Cells were induced with 0.1 μg/ml of AHT for 48 h and then infected with a CPAF wildtype (CPAF+) and CPAF mutant (CPAF-) strain, for another 24 h. Total cellular lysate...

Recombinant PI15 protein preparation. Recombinant PI15 proteins were purified from E. coli. Different concentrations of PI15 proteins were separated on a 12% PAGE gel and stained with Coomassie stain (left) or immunoblotted using an antibody against PI15 (right).

3D reconstruction of PI15-CPAF co-staining. Lateral rotation of the 3D projection around Y of C. trachomatis infected HeLa cells stained with CPAF (red channel), PI15 (green channel) and DNA is stained with DAPI (blue channel). Images were acquired in Z-stack on a Leica TCS SP5 confocal platform after fixing and reconstructed from the stacks using...

3D reconstruction from a coronal section of PI15-CPAF co-staining (The cell in entirety is seen in Video 1). Lateral rotation of the 3D projection around Y of C. trachomatis infected HeLa cells stained with CPAF (red channel), PI15 (green channel) and DNA is stained with DAPI (blue channel). Images were acquired in Z-stack on a Leica TCS SP5 confoc...

3D reconstruction from a coronal section of PI15-CPAF co-staining. Diagonal rotation of the 3D projection around Y of C. trachomatis infected HeLa cells stained with CPAF (red channel), PI15 (green channel) and DNA is stained with DAPI (blue channel). Images were acquired in Z-stack on a Leica TCS SP5 confocal platform after fixing and reconstructe...

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuol...

Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are two different species of betaherpesviruses that integrate into sub-telomeric ends of human chromosomes, for which different prevalence rates of integration have been reported. It has been demonstrated that integrated viral genome is stable and is fully retained. However, study of chromosomally integ...

Background: Oral candidiasis is an independent prognostic factor that can determine the progression of HIV infection. Widespread and repeated use of azole drugs has been implicated as the reason for the emerging fluconazole resistance in Candida strains. The K21 compound is an ethoxylated version of quaternary ammonium silane, with various antimicr...

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 protein...

Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that...

Raw data for quantitative analysis of C. trachomatis Cdu1-FLAG infectivity, Mcl-1 stabilization and apoptosis inhibition shown in Figure 3—figure supplement 2.DOI: http://dx.doi.org/10.7554/eLife.21465.013

Raw data for quantitative analysis of Mcl-1 stabilization in Cdu1-expressing HEK 293T cells shown in Figure 5.DOI: http://dx.doi.org/10.7554/eLife.21465.019

Raw data for quantitative analysis of Mcl-1 ring intensity around C. trachomatis pTet/Cdu1 inclusions uninduced and induced for Cdu1 overexpression shown in Figure 6—figure supplement 2.DOI: http://dx.doi.org/10.7554/eLife.21465.025

Raw data for quantitative analysis of Mcl-1 level in UO126-treated HeLa cells infected with C. trachomatis shown in Figure 1—figure supplement 1.DOI: http://dx.doi.org/10.7554/eLife.21465.005

Raw data for quantitative analysis of Mcl-1 stabilization, Mcl-1-ringed inclusion count and calculation of Mcl-1 ring intensity of HeLa cells infected with C. trachomatis wild type and Tn-cdu1 shown in Figure 6.DOI: http://dx.doi.org/10.7554/eLife.21465.022

Plasmids. Listed are all plasmids used in this study to transfect human cells or to transform E. coli or C. trachomatis. If not stated otherwise, constructs were cloned in this study using the oligo nucleotides listed in Supplementary file 4. DOI: http://dx.doi.org/10.7554/eLife.21465.035

Chlamydia trachomatis strains. Listed are all C. trachomatis strains used and generated in this study. All C. trachomatis strains generated by transformation and selection originate from the C. trachomatis LGV L2 (434) (ATCC VR-902B) strain. DOI: http://dx.doi.org/10.7554/eLife.21465.036

Raw data for quantitative analysis of Mcl-1 ubiquitination level shown in Figure 1.DOI: http://dx.doi.org/10.7554/eLife.21465.003

Raw data for analysis of relative enzymatic activity of Cdu1 in Ub-AMC hydrolysis assay shown in Figure 2—figure supplement 1.DOI: http://dx.doi.org/10.7554/eLife.21465.009

Raw data for quantitative analysis of apoptosis resistance analyzed by PARP cleavage and analysis of infectivity after IFNγ-treatment shown in Figure 7.DOI: http://dx.doi.org/10.7554/eLife.21465.027

Data collection and refinement statistics. X-ray crystallographic data collection and refinement statistics for the structure of Cdu1 (155-401). Data for the highest resolution shell are given in parentheses. a: Firedel’s mates were kept separately for calculation. DOI: http://dx.doi.org/10.7554/eLife.21465.034

Oligo nucleotides. Listed are all oligo nucleotides in 5’ → 3’ orientation used in this study. The oligo nucleotides were used for construct cloning, sequencing or southern hybridization as indicated in the comment column. DOI: http://dx.doi.org/10.7554/eLife.21465.037