The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7-Cyclin H Determines Individual Patterns of Transcription in Infected Cells.

The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.

Single-Cell RNA-Sequencing of RVFV Infection.

On the RNA level, viral infections are characterized by perturbations in the host cell transcriptome as well as the development of viral genetic information. Investigating the abundance and dynamic of RNA molecules can provide ample information to understand many aspects of the infection, from viral replication to pathogenesis. A key aspect therein is the resolution of the data, as infections are generally highly heterogeneous. Even in simple model systems such as cell lines, viral infections happen in a very asynchronous way. Quantifying RNAs at single-cell resolution can therefore substantially increase our understanding of these processes.Whereas measuring the RNA in bulk, that is, in samples containing thousands to hundreds of thousands of cells, is established and widely used since many years, methods for studying not only just a few different RNAs in individual cells became widely available only recently. Here, I outline and compare current concepts and methodologies for using single-cell RNA-sequencing to study virus infections. This covers sample preparation, cell preservation, biosafety considerations, and various experimental methods, with a special focus on the aspects that are important for studying virus infections. Since there is not "the one" method for doing single-cell RNA-sequencing, I will not provide a detailed protocol. Rather, this chapter should serve as a primer for getting started with single-cell RNA-sequencing experiments of virus infections and discusses the criteria that allow readers to choose the best procedures for their specific research question.

Pathogen dynamics and discovery of novel viruses and enzymes by deep nucleic acid sequencing of wastewater.

Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Protective role of the HSP90 inhibitor, STA-9090, in lungs of SARS-CoV-2-infected Syrian golden hamsters.

INTRODUCTION: The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyperinflammatory state generated on infection, reducing lung tissue pathology and inhibiting viral replication. Previous research has pointed to a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. METHODS: In this study, we investigated the effects of the potent HSP90 inhibitor Ganetespib (STA-9090) in vitro on alveolar epithelial cells and alveolar macrophages to characterise its effects on cell activation and viral replication. Additionally, the Syrian hamster animal model was used to evaluate its efficacy in controlling systemic inflammation and viral burden after infection. RESULTS: In vitro, STA-9090 reduced viral replication on alveolar epithelial cells in a dose-dependent manner and lowered significantly the expression of proinflammatory genes, in both alveolar epithelial cells and alveolar macrophages. In vivo, although no reduction in viral load was observed, administration of STA-9090 led to an overall improvement of the clinical condition of infected animals, with reduced oedema formation and lung tissue pathology. CONCLUSION: Altogether, we show that HSP90 inhibition could serve as a potential treatment option for moderate and severe cases of COVID-19.

Herpes simplex virus type 1 modifies the protein composition of extracellular vesicles to promote neurite outgrowth and neuroinfection.

The highly prevalent herpes simplex virus type 1 (HSV-1) causes a range of diseases, including cold sores, blinding keratitis, and life-threatening encephalitis. HSV-1 initially replicates in epithelial cells, enters the peripheral nervous system via neurites, and establishes lifelong infection in the neuronal cell bodies. Neurites are highly dynamic structures that grow or retract in response to attractive or repulsive cues, respectively. Here, we show that infection with HSV-1, but not with a mutant virus lacking glycoprotein G (gG), reduced the repulsive effect of epithelial cells on neurite outgrowth and facilitated HSV-1 invasion of neurons. HSV-1 gG was required and sufficient to induce neurite outgrowth by modifying the protein composition of extracellular vesicles, increasing the amount of neurotrophic and neuroprotective proteins, including galectin-1. Antibodies directed against galectin-1 neutralized the capacity of extracellular vesicles released from HSV-1-infected cells to promote neurite outgrowth. Our study provides new insights into the neurotropism of HSV-1 and identifies a viral protein that modifies the protein composition of extracellular vesicles to stimulate neurite outgrowth and invasion of the nervous system.IMPORTANCEHerpes simplex virus type 1 (HSV-1) must infect neurites (or nerve endings) to establish a chronic infection in neurons. Neurites are highly dynamic structures that retract or grow in the presence of repulsive or attractive proteins. Some of these proteins are released by epithelial cells in extracellular vesicles and act upon interaction with their receptor present on neurites. We show here that HSV-1 infection of epithelial cells modulated their effect on neurites, increasing neurite growth. Mechanistically, HSV-1 glycoprotein G (gG) modifies the protein composition of extracellular vesicles released by epithelial cells, increasing the amount of attractive proteins that enhance neurite outgrowth and facilitate neuronal infection. These results could inform of therapeutic strategies to block HSV-1 induction of neurite outgrowth and, thereby, neuronal infection.

Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19.

A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively.

Gut microbiota dysbiosis is associated with altered tryptophan metabolism and dysregulated inflammatory response in COVID-19.

The clinical course of COVID-19 is variable and often unpredictable. To test the hypothesis that disease progression and inflammatory responses associate with alterations in the microbiome and metabolome, we analyzed metagenome, metabolome, cytokine, and transcriptome profiles of repeated samples from hospitalized COVID-19 patients and uninfected controls, and leveraged clinical information and post-hoc confounder analysis. Severe COVID-19 was associated with a depletion of beneficial intestinal microbes, whereas oropharyngeal microbiota disturbance was mainly linked to antibiotic use. COVID-19 severity was also associated with enhanced plasma concentrations of kynurenine and reduced levels of several other tryptophan metabolites, lysophosphatidylcholines, and secondary bile acids. Moreover, reduced concentrations of various tryptophan metabolites were associated with depletion of Faecalibacterium, and tryptophan decrease and kynurenine increase were linked to enhanced production of inflammatory cytokines. Collectively, our study identifies correlated microbiome and metabolome alterations as a potential contributor to inflammatory dysregulation in severe COVID-19.

Proteomic and transcriptomic profiling of brainstem, cerebellum and olfactory tissues in early- and late-phase COVID-19.

Neurological symptoms, including cognitive impairment and fatigue, can occur in both the acute infection phase of coronavirus disease 2019 (COVID-19) and at later stages, yet the mechanisms that contribute to this remain unclear. Here we profiled single-nucleus transcriptomes and proteomes of brainstem tissue from deceased individuals at various stages of COVID-19. We detected an inflammatory type I interferon response in acute COVID-19 cases, which resolves in the late disease phase. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation, one neuronal with a direct focus on cranial nerve nuclei and a separate diffuse pattern affecting the whole brainstem. The latter reflects a bystander effect of the respiratory infection that spreads throughout the vascular unit and alters the transcriptional state of mainly oligodendrocytes, microglia and astrocytes, while alterations of the brainstem nuclei could reflect the connection of the immune system and the central nervous system via, for example, the vagus nerve. Our results indicate that even without persistence of severe acute respiratory syndrome coronavirus 2 in the central nervous system, local immune reactions are prevailing, potentially causing functional disturbances that contribute to neurological complications of COVID-19.

Herpesviruses mimic zygotic genome activation to promote viral replication.

DUX4 is a germline transcription factor and a master regulator of zygotic genome activation. During early embryogenesis, DUX4 is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In adult somatic cells, DUX4 expression is silenced and its activation in adult muscle cells causes the genetic disorder Facioscapulohumeral Muscular Dystrophy (FSHD). Here we show that herpesviruses from alpha-, beta- and gamma-herpesvirus subfamilies as well as papillomaviruses actively induce DUX4 expression to promote viral transcription and replication. We demonstrate that HSV-1 immediate early proteins directly induce expression of DUX4 and its target genes including endogenous retroelements, which mimics zygotic genome activation. We further show that DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for herpesvirus infection, since genetic depletion of DUX4 by CRISPR/Cas9 abrogates viral replication. Our results show that herpesviruses induce DUX4 expression and its downstream germline-specific genes and retroelements, thus mimicking an early embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.