Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7.

Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.

[Continuous SARS-CoV-2 monitoring in day care centers: A qualitative interview study on the experiences of childcare workers and parents with different self-sampling methods in the home environment]. / Kontinuierliche Überwachung von SARS-CoV-2 Infektionen in Kindertagesstätten: eine qualitative Interviewstudie über die Erfahrungen von Betreuer*innen und Eltern mit verschiedenen Testverfahren im häuslichen Umfeld.

BACKGROUND: Since the beginning of the corona pandemic in Germany in January 2020, day care centres (DCC) have faced the challenge of reliably detecting cases of SARS-CoV-2 infection as early and reliably as possible in order to avoid major outbreaks and closures. Conducting regular virological screening tests for asymptomatic DCC children and childcare workers requires a high level of acceptance among participants and should be as easy as possible to implement. The present study aimed to evaluate childcare workers' and parents' attitudes and experiences regarding the acceptance and feasibility of various screening methods. This assessment was conducted using additional qualitative interviews designed for home-based screening in the context of a screening study in DCCs. METHOD: From May to July 2021, childcare workers and parents of children in nine DCCs in Wuerzburg independently carried out screening tests for SARS-CoV-2 at home twice a week as part of the "Würzburg Child Care Study in the COVID-19 Pandemic 2.0". The participants were offered two self-sampling methods (mouth-rinsing fluid for pooled PCR tests and/or nasal rapid antigen self-tests). Before and after the 12-week test phase, telephone interviews were conducted with a selected sample of childcare workers and parents in order to ascertain initial attitudes and further experiences with the two self-sampling methods and their implementation. The interviews were fully transcribed for analysis and subjected to a qualitative content analysis according to Kuckartz. RESULTS: Of the 1,026 eligible participants, 591 individuals, including 139 childcare workers and the parents of 452 children, agreed to take part in the screening tests. A total of 49 interviews were conducted with a specifically selected sample (20 before the start of the test, 29 after the end of the test). In the qualitative content analysis, three overarching topics emerged: 'aspects of the test performance', 'aspects of the perception of safety' and 'aspects of the testing in children'. Regardless of the fact that the various test methods and test features were perceived very differently, conducting the tests at home was found to be feasible. DISCUSSION: The differentiated insights into the participants' perspectives provide valuable information about factors that influence the acceptance of self-testing. These should be taken into account before such a measure is introduced in DCCs if necessary. CONCLUSION: The assessment of test procedures is strongly influenced by individual preferences. Test concepts should be implemented with as little time and organisational effort as possible in order to promote willingness to participate. Clear study information and quick feedback on test results can enhance the sense of security among parents and childcare workers.

Heterologous and homologous COVID-19 mRNA vaccination schemes for induction of basic immunity show similar immunogenicity regarding long-term spike-specific cellular immunity in healthcare workers.

Healthcare workers (HCWs) are recommended to receive at least three spike-antigen exposures to generate basic immunity and to mediate herd protection of vulnerable patients. So far, less attention has been put on the cellular immune response induced by homologous (three BTN162b2mRNA doses) or heterologous (mRNA-1273 as third dose building on two BTN162bmRNA doses) and the immunological impact of breakthrough infections (BTIs). Therefore, in 356 vaccinated HCWs with or without BTIs the Anti-SARS-CoV-2-Spike-IgG concentrations and avidities and B- and T-cell-reactivity against SARS-CoV-2-Spike-S1- and Nucleocapsid-antigens were assessed with Interferon-gamma-ELISpot and by flow-cytometry. HCWs who had hybrid immunity due to BTIs exhibited strong T-cell-reactivity against the Spike-S1-antigen. A lasso regression model revealed a significant reduction in T-cell immune responses among smokers (p < 0.0001), with less significant impact observed for age, sex, heterologous vaccination, body-mass-index, Anti-Nucleocapsid T-cell reactivity, days since last COVID-19-immunization, and Anti-SARS-CoV-2-Spike-IgG. Although subgroup analysis revealed higher Anti-SARS-CoV-2-Spike-IgG after heterologous vaccination, similar cellular reactivity and percentages of Spike-reactive T- and B-cells were found between homologous and heterologous vaccination. Anti-SARS-CoV-2-Spike-IgG concentrations and avidity significantly correlated with activated T-cells. CD4 + and CD8 + responses correlated with each other. A strong long-term cellular immune response should be considered as baseline for recommendations of booster doses in HCWs with prioritization of smokers. HCWs presented significant T-cellular reactivity towards Spike-S1-antigen with particularly strong responses in hybrid immunized HCWs who had BTIs. HCWs without BTI presented similar percentages of Spike-specific B- and T-cells between homologous or heterologous vaccination indicating similar immunogenicity for both mRNA vaccines, BNT162b2mRNA and mRNA-1273.

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.

Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5 min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning.

Human leucocyte antigen class I (HLA-I) molecules play a central role for both NK and T-cell responses that prevent serious human cytomegalovirus (HCMV) disease. To create opportunities for viral spread, several HCMV-encoded immunoevasins employ diverse strategies to target HLA-I. Among these, the glycoprotein US10 is so far insufficiently studied. While it was reported that US10 interferes with HLA-G expression, its ability to manipulate classical HLA-I antigen presentation remains unknown. In this study, we demonstrate that US10 recognizes and binds to all HLA-I (HLA-A, -B, -C, -E, -G) heavy chains. Additionally, impaired recruitment of HLA-I to the peptide loading complex was observed. Notably, the associated effects varied significantly dependending on HLA-I genotype and allotype: (i) HLA-A molecules evaded downregulation by US10, (ii) tapasin-dependent HLA-B molecules showed impaired maturation and cell surface expression, and (iii) ß2m-assembled HLA-C, in particular HLA-C*05:01 and -C*12:03, and HLA-G were strongly retained in complex with US10 in the endoplasmic reticulum. These genotype-specific effects on HLA-I were confirmed through unbiased HLA-I ligandome analyses. Furthermore, in HCMV-infected fibroblasts inhibition of overlapping US10 and US11 transcription had little effect on HLA-A, but induced HLA-B antigen presentation. Thus, the US10-mediated impact on HLA-I results in multiple geno- and allotypic effects in a so far unparalleled and multimodal manner.

During a viral infection, the immune system must discriminate between healthy and infected cells to selectively kill infected cells. Healthy cells have different types of molecules known collectively as HLA-I on their surface. These molecules present small fragments of proteins from the cell, called antigens, to patrolling immune cells, known as CTLs or natural killer cells. While CTLs ignore antigens from human proteins (which indicate the cell is healthy), they can bind to and recognize antigens from viral proteins, which triggers them to activate immune responses that kill the infected cell. However, some viruses can prevent infected cells from presenting HLA-I molecules on their surfaces as a strategy to evade the immune system. Natural killer cells have evolved to overcome this challenge. They bind to the HLA-I molecules themselves, which causes them to remain inactive. However, if the HLA-I molecules are missing, the NK cells can more easily switch on and kill the target cell. The human cytomegalovirus is a common virus that causes lifelong infection in humans. Although it rarely causes illness in healthy individuals, it can be life-threatening to newborn babies and for individuals with weakened immune systems. One human cytomegalovirus protein known as US10 was previously found to bind to HLA-I without reducing the levels of these molecules on the surface of the cell. However, its precise role remained unclear. Gerke et al. used several biochemical and cell biology approaches to investigate whether US10 manipulates the quality of the three types of HLA-I, which could impact both CTL and NK cell recognition. The experiments showed that US10 acted differently on the various kinds of HLA-I. To one type, it bound strongly within the cell and prevented it from reaching the surface. US10 also prevented another type of HLA-I from maturing properly and presenting antigens but did not affect the third type of HLA-I. These findings suggest that US10 interferes with the ability of different HLA-I types to present antigens in specific ways. Further research is needed to measure how US10 activity affects immune cells, which may ultimately aid the development of new therapies against human cytomegalovirus and other similar viruses.

Human cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

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.

Impaired T cells and "memory-like" NK-cell reconstitution is linked to late-onset HCMV reactivation after letermovir cessation.

ABSTRACT: Allogeneic hematopoietic stem cell transplantation (alloSCT) is the only cure for many hematologic malignancies. However, alloSCT recipients are susceptible to opportunistic pathogens, such as human cytomegalovirus (HCMV). Letermovir prophylaxis has revolutionized HCMV management, but the challenge of late HCMV reactivations has emerged. Immunological surrogates of clinically significant HCMV infection (csCMVi) after discontinuation of letermovir remain to be defined. Therefore, we studied natural killer (NK)-cell reconstitution along with the global and HCMV pp65-specific T-cell repertoire of 24 alloSCT recipients at 7 time points before (day +90) and after (days +120-270) cessation of letermovir prophylaxis. Patients who experienced csCMVi had lower counts of IFN-γ+ HCMV-specific CD4+ and CD8+ T cells than HCMV controllers. Furthermore, patients with csCMVi displayed late impairment of NK-cell reconstitution, especially suppression of "memory-like" CD159c+CD56dim NK-cell counts that preceded csCMVi events in most patients. Moreover, several surrogates of immune reconstitution were associated with the severity of HCMV manifestation, with patients suffering from HCMV end-organ disease and/or refractory HCMV infection harboring least HCMV-specific T cells and "memory-like" NK cells. Altogether, our findings establish an association of delayed or insufficient proliferation of both HCMV-specific T cells and "memory-like" NK cells with csCMVi and the severity of HCMV manifestations after discontinuation of letermovir prophylaxis.

Identification of novel canonical and cryptic HCMV-specific T-cell epitopes for HLA-A∗03 and HLA-B∗15 via peptide-PRISM.

ABSTRACT: Human cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. 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 by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03-restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15-restricted immunogenic peptide, with a specific interferon gamma-positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03-restricted and 3 HLA-B∗15-restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets.

SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9.

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

The HSV-1 ICP22 protein selectively impairs histone repositioning upon Pol II transcription downstream of genes.

Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-ß Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

The relationship between mental health, sleep quality and the immunogenicity of COVID-19 vaccinations.

Sleep modulates the immune response, and sleep loss can reduce vaccine immunogenicity; vice versa, immune responses impact sleep. We aimed to investigate the influence of mental health and sleep quality on the immunogenicity of COVID-19 vaccinations and, conversely, of COVID-19 vaccinations on sleep quality. The prospective CoVacSer study monitored mental health, sleep quality and Anti-SARS-CoV-2-Spike IgG titres in a cohort of 1082 healthcare workers from 29 September 2021 to 19 December 2022. Questionnaires and blood samples were collected before, 14 days, and 3 months after the third COVID-19 vaccination, as well as in 154 participants before and 14 days after the fourth COVID-19 vaccination. Healthcare workers with psychiatric disorders had slightly lower Anti-SARS-CoV-2-Spike IgG levels before the third COVID-19 vaccination. However, this effect was mediated by higher median age and body mass index in this subgroup. Antibody titres following the third and fourth COVID-19 vaccinations ("booster vaccinations") were not significantly different between subgroups with and without psychiatric disorders. Sleep quality did not affect the humoral immunogenicity of the COVID-19 vaccinations. Moreover, the COVID-19 vaccinations did not impact self-reported sleep quality. Our data suggest that in a working population neither mental health nor sleep quality relevantly impact the immunogenicity of COVID-19 vaccinations, and that COVID-19 vaccinations do not cause a sustained deterioration of sleep, suggesting that they are not a precipitating factor for insomnia. The findings from this large-scale real-life cohort study will inform clinical practice regarding the recommendation of COVID-19 booster vaccinations for individuals with mental health and sleep problems.

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.

CD4+ T cells are the major predictor of HCMV control in allogeneic stem cell transplant recipients on letermovir prophylaxis.

Introduction: Human cytomegalovirus (HCMV) causes significant morbidity and mortality in allogeneic stem cell transplant (alloSCT) recipients. Recently, antiviral letermovir prophylaxis during the first 100 days after alloSCT replaced PCR-guided preemptive therapy as the primary standard of care for HCMV reactivations. Here, we compared NK-cell and T-cell reconstitution in alloSCT recipients receiving preemptive therapy or letermovir prophylaxis in order to identify potential biomarkers predicting prolonged and symptomatic HCMV reactivation. Methods: To that end, the NK-cell and T-cell repertoire of alloSCT recipients managed with preemptive therapy (n=32) or letermovir prophylaxis (n=24) was characterized by flow cytometry on days +30, +60, +90 and +120 after alloSCT. Additionally, background-corrected HCMV-specific T-helper (CD4+IFNγ+) and cytotoxic (CD8+IFNγ+CD107a+) T cells were quantified after pp65 stimulation. Results: Compared to preemptive therapy, letermovir prophylaxis prevented HCMV reactivation and decreased HCMV peak viral loads until days +120 and +365. Letermovir prophylaxis resulted in decreased T-cell numbers but increased NK-cell numbers. Interestingly, despite the inhibition of HCMV, we found high numbers of "memory-like" (CD56dimFcεRIγ- and/or CD159c+) NK cells and an expansion of HCMV-specific CD4+ and CD8+ T cells in letermovir recipients. We further compared immunological readouts in patients on letermovir prophylaxis with non/short-term HCMV reactivation (NSTR) and prolonged/symptomatic HCMV reactivation (long-term HCMV reactivation, LTR). Median HCMV-specific CD4+ T-cell frequencies were significantly higher in NSTR patients (day +60, 0.35 % vs. 0.00 % CD4+IFNγ+/CD4+ cells, p=0.018) than in patients with LTR, whereas patients with LTR had significantly higher median regulatory T-cell (Treg) frequencies (day +90, 2.2 % vs. 6.2 % CD4+CD25+CD127dim/CD4+ cells, p=0.019). ROC analysis confirmed low HCMV specific CD4+ (AUC on day +60: 0.813, p=0.019) and high Treg frequencies (AUC on day +90: 0.847, p=0.021) as significant predictors of prolonged and symptomatic HCMV reactivation. Discussion: Taken together, letermovir prophylaxis delays HCMV reactivation and alters NK- and T-cell reconstitution. High numbers of HCMV-specific CD4+ T cells and low numbers of Tregs seem to be pivotal to suppress post-alloSCT HCMV reactivation during letermovir prophylaxis. Administration of more advanced immunoassays that include Treg signature cytokines might contribute to the identification of patients at high-risk for long-term and symptomatic HCMV reactivation who might benefit from prolonged administration of letermovir.

HSV-1 Infection Induces a Downstream Shift of Promoter-Proximal Pausing for Host Genes.

Herpes simplex virus 1 (HSV-1) infection exerts a profound shutoff of host gene expression at multiple levels. Recently, HSV-1 infection was reported to also impact promoter-proximal RNA polymerase II (Pol II) pausing, a key step in the eukaryotic transcription cycle, with decreased and increased Pol II pausing observed for activated and repressed genes, respectively. Here, we demonstrate that HSV-1 infection induces more complex alterations in promoter-proximal pausing than previously suspected for the vast majority of cellular genes. While pausing is generally retained, it is shifted to more downstream and less well-positioned sites for most host genes. The downstream shift of Pol II pausing was established between 1.5 and 3 h of infection, remained stable until at least 6 hours postinfection, and was observed in the absence of ICP22. The shift in Pol II pausing does not result from alternative de novo transcription initiation at downstream sites or read-in transcription originating from disruption of transcription termination of upstream genes. The use of downstream secondary pause sites associated with +1 nucleosomes was previously observed upon negative elongation factor (NELF) depletion. However, downstream shifts of Pol II pausing in HSV-1 infection were much more pronounced than observed upon NELF depletion. Thus, our study reveals a novel aspect in which HSV-1 infection fundamentally reshapes host transcriptional processes, providing new insights into the regulation of promoter-proximal Pol II pausing in eukaryotic cells. IMPORTANCE This study provides a genome-wide analysis of changes in promoter-proximal polymerase II (Pol II) pausing on host genes induced by HSV-1 infection. It shows that standard measures of pausing, i.e., pausing indices, do not properly capture the complex and unsuspected alterations in Pol II pausing occurring in HSV-1 infection. Instead of a reduction of pausing with increased elongation, as suggested by pausing index analysis, HSV-1 infection leads to a shift of pausing to downstream and less well-positioned sites than in uninfected cells for the majority of host genes. Thus, HSV-1 infection fundamentally reshapes a key regulatory step at the beginning of the host transcriptional cycle on a genome-wide scale.

Influencing factors of anti-SARS-CoV-2-spike-IgG antibody titers in healthcare workers: A cross-section study.

Against the background of the current COVID-19 infection dynamics with its rapid spread of SARS-CoV-2 variants of concern (VOC), the immunity and the vaccine prevention of healthcare workers (HCWs) against SARS-CoV-2 continues to be of high importance. This observational cross-section study assesses factors influencing the level of anti-SARS-CoV-2-spike IgG after SARS-CoV-2 infection or vaccination. One thousand seven hundred and fifty HCWs were recruited meeting the following inclusion criteria: age ≥18 years, PCR-confirmed SARS-CoV-2 infection convalescence and/or at least one dose of COVID-19 vaccination. anti-SARS-CoV-2-spike IgG titers were determined by SERION ELISA agile SARS-CoV-2 IgG. Mean anti-SARS-CoV-2-spike IgG levels increased significantly by number of COVID-19 vaccinations (92.2 BAU/ml for single, 140.9 BAU/ml for twice and 1144.3 BAU/ml for threefold vaccination). Hybrid COVID-19 immunized respondents (after infection and vaccination) had significantly higher antibody titers compared with convalescent only HCWs. Anti-SARS-CoV-2-spike IgG titers declined significantly with time after the second vaccination. Smoking and high age were associated with lower titers. Both recovered and vaccinated HCWs presented a predominantly good humoral immune response. Smoking and higher age limited the humoral SARS-CoV-2 immunity, adding to the risk of severe infections within this already health impaired collective.