570 Biowissenschaften; Biologie
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Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner.
A fine balance of regulatory (T\(_{reg}\)) and conventional CD4\(^+\) T cells (T\(_{conv}\)) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the T\(_{reg}\)/T\(_{conv}\) balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse T\(_{reg}\) and T\(_{conv}\) with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C\(_{16}\)-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into T\(_{reg}\) and T\(_{conv}\) reflect differences in the ceramide content of cellular membranes.
MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards naïve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.
Measles is an ancient disease with historical records as early as the 9th century.
Extensive study as well as advances in scientific knowledge of virology have led to
identification of the viral pathogen and subsequent development of an effective vaccine
leading to global efforts towards measles elimination. In 2018, around 140,000 deaths were
reported due to measles with incomplete vaccine coverage being one of the leading causes
of resurgence. Measles is highly contagious and often regarded as a childhood illness.
However, measles is associated with a number of complications and persistent infections
like subacute sclerosing panencephalitis (SSPE), which have brought into focus the need
for specific anti-viral therapies.
The aim of this study was to target host and viral factors to optimize anti-measles virus
therapy. Our approach was to test a panel of compounds known to inhibit host cell
functions or viral factors for their antiviral effect on measles replication. Primary human
lymphocytes, persistently infected NT2 cells and post-mitotic neurons were used as in vitro
model systems of acute, persistent and neuronal infection respectively to test the inhibitors.
Using the inhibitors Ceranib-2 and SKI-II to target the sphingolipid metabolism enzymes
acid ceramidase and sphingosine kinase in infected human primary lymphocytes, we
observed a decreased protein translational capacity mediated by mTORC1, EIF4E and
ribosomal protein S6 phosphorylation that probably contributes to the antiviral effect. In
the persistently infected neural NT2 cells and post-mitotic neurons derived from LUHMES
cells, we observed effective infection inhibition and viral clearance upon treatment with a
small non-nucleoside inhibitor (ERDRP-0519) specifically targeting the Morbillivirus
large polymerase. Other inhibitors such as Ribavirin and Favipiravir were less effective. To
conclude, 1) we identified a mTOR associated protein translation axis associated with the
sphingolipid metabolism, which affects measles virus replication and 2) In vitro
persistently infected neuronal and post-mitotic neuron models were successfully used as a
rapid method to test antivirals against measles virus.
ITN—VIROINF: Understanding (harmful) virus-host interactions by linking virology and bioinformatics
(2021)
Many recent studies highlight the fundamental importance of viruses. Besides their important role as human and animal pathogens, their beneficial, commensal or harmful functions are poorly understood. By developing and applying tailored bioinformatical tools in important virological models, the Marie Skłodowska-Curie Initiative International Training Network VIROINF will provide a better understanding of viruses and the interaction with their hosts. This will open the door to validate methods of improving viral growth, morphogenesis and development, as well as to control strategies against unwanted microorganisms. The key feature of VIROINF is its interdisciplinary nature, which brings together virologists and bioinformaticians to achieve common goals.
Transcription describes the process of converting the information contained in DNA into RNA. Although, tremendous progress has been made in recent decades to uncover this complex mechanism, it is still not fully understood. Given the advances and reduction in cost of high-throughput sequencing experiments, more and more data have been generated to help elucidating this complex process. Importantly, these sequencing experiments produce massive amounts of data that are incomprehensible in their raw form for humans. Further, sequencing techniques are not always 100% accurate and are subject to a certain degree of variability and, in special cases, they might introduce technical artifacts. Thus, computational and statistical methods are indispensable to uncover the information buried in these datasets.
In this thesis, I worked with multiple high throughput datasets from herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. During the last decade, it has became clear that a gene might not have a single, but multiple sites at which transcription initiates. These multiple transcription start sites (TiSS) demonstrated to have regulatory effects on the gene itself depending on which TiSS is used. Specialized experimental approaches were developed to help identify TiSS (TiSS-profiling). In order to facilitate the identification of all potential TiSS that are used for cell type- and condition-specific transcription, I developed the tool iTiSS. By using a new general enrichment-based approach to predict TiSS, iTiSS proved to be applicable in integrated studies and made it less prone to false positives compared to other TiSS-calling tools. Another improvement in recent years was made in metabolic labeling experiments such as SLAM-seq. Here, they removed the time consuming and laborious step of physically separating new from old RNA in the samples. This was achieved by inducing specific nucleotide conversions in newly synthesized RNA that are later visible in the data. Consequently, the separation of new and old RNA is now done computationally and, hence, tools are needed that accurately quantify these fold-changes. My second tool that I developed, called GRAND-SLAM proved to be capable to accomplish this task and outperform competing programs. As both of my tools, iTiSS and GRAND-SLAM are not specifically tailored to my own goals, but could also facilitate the research of other groups in this field, I made them publicly available on GitHub.
I applied my tools to datasets generated in our lab as well as to publicly available data sets from HSV-1 and HCMV, respectively. For HSV-1, I was able to predict and validate TiSS with nucleotide precision using iTiSS. This has lead to the most comprehensive annotation for HSV-1 to date, which now serves as the fundamental basis of any future transcriptomic research on HSV-1. By combining both my tools, I was further able to uncover parts of the highly complex gene kinetics in HCMV and to resolve the limitations caused by the densely packed genome of HCMV.
With the ever-increasing advances in sequencing techniques and their decrease in cost, the amounts of data produced will continue to rise massively in the future. Additionally, more and more specialized omics approaches are appearing, calling for new tools to leverage their full information potential. Consequently, it has become apparent that specialized computational tools such as iTiSS and GRAND-SLAM are needed and will become an essential and indispensable part of the analysis.
Lack of acid sphingomyelinase (ASM) activity, either through genetic deficiency or through pharmacological inhibition, is linked with increased activity and frequency of Foxp3+ regulatory T cells (Treg) among cluster of differentiation (CD) 4+ T cells in mice in vivo and in vitro1. Thus, pharmacological blockade of ASM activity, which catalyzes the cleavage of sphingomyelin to ceramide and phosphocholine, might be used as a new therapeutic mechanism to correct numeric and/ or functional Treg de-ficiencies in diseases like multiple sclerosis or major depression.
In the present study, the effect of pharmacological inhibition of ASM in humans, in vitro and in vivo, was analyzed. In the in vitro experiments, peripheral blood mono-nuclear cells (PBMC) of healthy human blood donors were treated with two widely prescribed antidepressants with high (sertraline, Ser) or low (citalopram, Cit) capaci-ty to inhibit ASM activity. Similar to the findings in mice an increase in the frequency of Treg among human CD4+ T cells upon inhibition of ASM activity was observed. For the analysis in vivo, a prospective study of the composition of the CD4+ T cell com-partment of patients treated for major depression was done. The data show that pharmacological inhibition of ASM activity was superior to antidepressants with little or no ASM-inhibitory activity in increasing CD45RA- CD25high effector Treg (efTreg) frequencies among CD4+ T cells to normal levels. Independently of ASM inhibition, correlating the data with the clinical response, i.e. improvement of the Hamilton rat-ing scale for depression (HAMD) by at least 50 per cent (%) after four weeks of treatment, it was found that an increase in efTreg frequencies among CD4+ cells dur-ing the first week of treatment identified patients with a clinical response.
Regarding the underlying mechanism, it could be found that the positive effect of ASM inhibition on Treg required CD28 co-stimulation suggesting that enhanced CD28 co-stimulation was the driver of the observed increase in the frequency of Treg among human CD4+ T cells. Inhibition of ASM activity was further associated with changes in the expression and shuttling of CTLA-4, a key inhibitory molecule ex-pressed by Treg, between cellular compartments but the suppressive activity of CTLA-4 through its transendocytosis activity was unaffected by the inhibition of ASM activity.
In summary, the frequency of (effector) Treg among CD4+ T cells in mice and in hu-mans is increased after inhibition of ASM activity suggesting that ASM blockade might beneficially modulate autoimmune diseases and depression-promoting in-flammation.
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.
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 all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research.
The respiratory system is amongst the most important compartments in the human body. Due to its connection to the external environment, it is one of the most common portals of pathogen entry. Airborne pathogens like measles virus (MV) carried in liquid droplets exhaled from the infected individuals via a cough or sneeze enter the body from the upper respiratory tract and travel down to the lower respiratory tract and reach the alveoli. There, pathogens are captured by the resident dendritic cells (DCs) or macrophages and brought to the lymph node where immune responses or, as in case of MV, dissemination via the hematopoietic cell compartment are initiated. Basic mechanisms governing MV exit from the respiratory tract, especially virus transmission from infected immune cells to the epithelial cells have not been fully addressed before. Considering the importance of these factors in the viral spread, a complex close-to-in-vivo 3D human respiratory tract model was generated. This model was established using de-cellularized porcine intestine tissue as a biological scaffold and H358 cells as targets for infection. The scaffold was embedded with fibroblast cells, and later on, an endothelial cell layer seeded at the basolateral side. This provided an environment resembling the respiratory tract where MV infected DCs had to transmigrate through the collagen scaffold and transmit the virus to epithelial cells in a Nectin-4 dependent manner. For viral transmission, the access of infected DCs to the recipient epithelial cells is an essential prerequisite and therefore, this important factor which is reflected by cell migration was analyzed in this 3D system.
The enhanced motility of specifically MV-infected DCs in the 3D models was observed, which occurred independently of factors released from the other cell types in the models. Enhanced motility of infected DCs in 3D collagen matrices suggested infection-induced cytoskeletal remodeling, as also verified by detection of cytoskeletal polarization, uropod formation. This enforced migration was sensitive to ROCK inhibition revealing that MV infection induces an amoeboid migration mode in DCs. In support of this, the formation of podosome structures and filopodia, as well as their activity, were reduced in infected DCs and retained in their uninfected siblings. Differential migration modes of uninfected and infected DCs did not cause differential maturation, which was found to be identical for both populations. As an underlying mechanism driving this enforced migration, the role of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) was studied in MV-exposed cultures. It was shown in this thesis that MV-infection increased S1P production, and this was identified as a contributing factor as inhibition sphingosine kinase activity abolished enforced migration of MV-infected DCs. These findings revealed that MV infection induces a fast push-and-squeeze amoeboid mode of migration, which is supported by SphK/S1P axis. However, this push-and-squeeze amoeboid migration mode did not prevent the transendothelial migration of MV-infected DCs.
Altogether, this 3D system has been proven to be a suitable model to study specific parameters of mechanisms involved in infections in an in vivo-like conditions.