Role of SARS-CoV-2 in Altering the RNA-Binding Protein and miRNA-Directed Post-Transcriptional Regulatory Networks in Humans

The outbreak of a novel coronavirus SARS-CoV-2 responsible for the COVID-19 pandemic has caused a worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2-mediated alterations on post-transcriptional gene regulations across human tissues remain elusive. In this study, we analyzed publicly available genomic datasets to systematically dissect the crosstalk and dysregulation of the human post-transcriptional regulatory networks governed by RNA-binding proteins (RBPs) and micro-RNAs (miRs) due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2-encoded proteins directly interacted with 51 human RBPs, of which the majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed a functional analysis of differentially expressed genes in mock-treated versus SARS-CoV-2-infected lung cells that revealed enrichment for the immune response, cytokine-mediated signaling, and metabolism-associated genes. This study also characterized the alternative splicing events in SARS-CoV-2-infected cells compared to the control, demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to the viral infection. A motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed the enrichment for RBPs such as SRSFs, PCBPs, ELAVs, and HNRNPs, suggesting the sponging of RBPs by the SARS-CoV-2 genome. A similar analysis to study the interactions of miRs with SARS-CoV-2 revealed functionally important miRs that were highly expressed in immune cells, suggesting that these interactions may contribute to the progression of the viral infection and modulate the host immune response across other human tissues. Given the need to understand the interactions of SARS-CoV-2 with key post-transcriptional regulators in the human genome, this study provided a systematic computational analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs across tissue types during a SARS-CoV-2 infection

The impact of influenza on the behaviour of lung basal cells

Influenza is the cause of 5 million infections globally, resulting in around 500,000 deaths each year. Despite targeted anti-viral drugs and vaccines, influenza viruses are poorly controlled and pose a particular threat to those who suffer chronic diseases. During infection, virus and immune-mediated damage destroys the airway structure needed for gas exchange and repairing this damage is essential for survival. Lung repair is driven by activation of progenitor cells, but how they carry out this repair is not well understood. Lung progenitors are difficult to study as they are a rare cell type in the lung, and the protocols for lung digest are not optimised for recovery of progenitors. We developed a new protocol for increasing the yield of progenitors from mouse lung tissue and used published transcriptomics data sets to identify progenitor cells in silico. We aimed to understand how the composition of the lung was changed during damage, so we infected mice with influenza A virus and studied how the behaviour of the epithelial progenitors changed. We found the composition of the lung changes during the peak and recovery phases of influenza. This change in composition correlates with increased activation and proliferation of progenitors and we show that this is accompanied by a change in cell metabolism, particularly an increase in glycolysis. We found that upper airway basal cells rely on IL-10 signalling to initiate this activation, and we suggest that the immunoregulatory response is necessary to initiate basal cell activation. We considered that lung basal cells might be changed by direct interaction with influenza viruses and using novel, fluorescent influenza A virus, we show that a subset of lung basal cells are directly infected by influenza virus but survive. Direct infection triggers an increase in stress, and further increases in glycolytic metabolism. We suggest this metabolic adaption of epithelial progenitors is a critical step in initiating lung repair after damage, and the stress seen in directly infected cells could result in improper dysplastic repair. Our data support the proposal that new treatments for respiratory diseases could consider targeting mechanisms of repair. Improving how the host repairs damage could not only be effective alongside drugs and vaccines in treating acute infections like influenza, but also in resolving chronic inflammatory such as COPD

Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of great concern to human health. The objective of this study was to identify miRNAs associated with AIV infections in chickens.</p> <p>Results</p> <p>Total RNAs were isolated from lung and trachea of low pathogenic H5N3 infected and non-infected SPF chickens at 4 days post-infection. A total of 278,398 and 340,726 reads were obtained from lung and trachea, respectively. And 377 miRNAs were detected in lungs and 149 in tracheae from a total of 474 distinct chicken miRNAs available at the miRBase, respectively. Seventy-three and thirty-six miRNAs were differentially expressed between infected and non-infected chickens in lungs and tracheae, respectively. There were more miRNAs highly expressed in non-infected tissues than in infected tissues. Interestingly, some of these differentially expressed miRNAs, including miR-146, have been previously reported to be associated with immune-related signal pathways in mammals.</p> <p>Conclusion</p> <p>To our knowledge, this is the first study on miRNA gene expression in AIV infected chickens using a deep sequencing approach. During AIV infection, many host miRNAs were differentially regulated, supporting the hypothesis that certain miRNAs might be essential in the host-pathogen interactions. Elucidation of the mechanism of these miRNAs on the regulation of host-AIV interaction will lead to the development of new control strategies to prevent or treat AIV infections in poultry.</p

RNA species in the host-pathogen dynamics during Legionella infection of human macrophages

Legionella pneumophila (L.p.) is a gram-negative, intracellular pathogen and a common cause of severe community-acquired pneumonia. In humans, L.p. replicates primarily within alveolar macrophages. It manipulates vital host cell functions such as vesicle trafficking and gene expression by the secretion of over 300 effector proteins into the host cell cytosol. Thus, L.p. modifies its host cell to promote its own replication. An unbiased and global analysis of the molecular changes and biological processes that are associated with bacterial infections of human cells can provide new insights into host-pathogen interactions. Therefore, one goal of this study was to characterize expression changes of different RNA species in response to infection with L.p. in human primary blood-derived macrophages (BDMs) or differentiated THP-1 cells. This work is structured into two parts: (1) a functional study on how miRNA manipulations can alter L.p. replication in macrophages and (2) an in depth analysis of transcriptomic events in host and pathogen during infection. (1) In the last few decades, miRNAs have been established as critical modulators of immune function. Therefore, one aim of this study was to identify the miRNA profile of L.p.-infected macrophages and to determine the functional impact of a miRNA manipulation on L.p. replication. BDMs of healthy donors were infected with L.p. strain Corby. Small RNA sequencing revealed the miRNA profile in BDMs following L.p. infection. An upregulation of miR-146a and miR-155, as well as downregulation of miR-221 and miR-125b was validated by qPCR in macrophages. miRNA regulation in response to infection seems to be due to transcriptional regulation of miRNA promoters, since the histone acetylation levels at the promoter and the pri-miR expression correlated with the miRNA expression upon L.p.- infection. Overexpression and knock down experiments of miR-125b, miR-221 and miR-579 in combination were performed for functional characterization and showed an influence of all three miRNAs on bacterial replication. A SILAC approach revealed the protein MX1 as downregulated following simultaneous overexpression of all three miRNAs. MX1 is an interferon-induced GTP-binding protein important for antiviral defence. As shown by validation experiments, MX1 knockdown in macrophages led to an increased replication of L.p., as seen following overexpression of the miRNAs. Since in silico analysis predicted no binding sites for either miRNA in the 3’UTR of MX1, Ingenuity pathway analysis was performed to find the linking molecules. DDX58 (RIG-I), a sensor for cytosolic RNA, was validated as a target for miR-221, while the tumour suppressor TP53 was shown to be targeted by miR-125b via luciferase reporter assays. An siRNA-mediated knockdown of both, TP53 and DDX58, respectively, led to an enhanced replication of L.p. in macrophages. Thus, DDX58 and TP53 were validated as linking molecules between the three miRNAs and MX1. Additionally, the aforementioned SILAC approach revealed a downregulation of LGALS8 which was later validated as a target of miR-579. LGALS8 is a cytosolic lectin which binds carbohydrates and localizes to damaged vesicles. Knockdown of LGALS8 enhanced intracellular replication in macrophages. Thus, MX1 and LGALS8 were identified as targets of the three miRNAs (miR-125b, miR-221, miR-579) and to be responsible for the restriction of L.p. replication within human macrophages. (2) The transcriptional profile of L.p. during the course of infection in human macrophages was next to be established. Dual RNA-Sequencing was performed to determine the regulation of coding and non-coding RNA species during the course of infection of both, host and pathogen, simultaneously. After adaptation and optimization of existing protocols, macrophages were infected using a GFP-expressing L.p. strain Corby. To separate infected cells (gfp+) from the non-invaded bystander cells (gfp-), flow cytometry sorting was performed. Furthermore, Pam3CSK4 was used to generate TLR2-activated cells. RNA from all different samples, and also RNA from cultivated Legionella, was sequenced. Differential gene expression analysis was performed using DESeq2 resulting in 4,144 differentially expressed human genes (across multiple conditions) and 2,707 differentially expressed Legionella genes (across two time points). The DESeq analysis of the separated RNA fractions from host cells revealed differentially expressed mRNAs (3,504), lncRNAs (495), and miRNAs (145). 1,128 differentially expressed genes were exclusively significantly regulated in invaded cells (gfp+ at 8 and 16 h). Some of these were validated via qPCR including BCL10, SOD1, IRS1, CYR61, ATG5, RND3 and JUN. In addition, the simultaneous upregulation of the genes ZFAND2A and HSPA1 in the bystander and in Legionella-invaded cells was validated. The analysis of the bacterial mRNAs revealed a switch of gene usage, i.e. inverse regulation at 8 and 16 h post infection. This switch included genes which are involved in iron metabolism, stress response, glycolysis and lipid biosynthesis. Hence, differentially expressed genes within different growth phases of the infection cycle were identified. This dataset is the first of its kind to cover a respiratory pathogen. The dual RNA-Sequencing performed in this study provides data to encapsulate the RNA landscape of coding and non-coding RNAs in pathogen and host. In summary, the results have deepened our insight into the infection process and the molecular interaction of L.p. and its host cells and will help to understand the complex interplay between host and pathogen by allowing for the in silico re-construction of an RNA interaction network. Furthermore, the present study will help to establish potential new candidates for diagnosis and therapy

RNA species in the host-pathogen dynamics during Legionella infection of human macrophages

Legionella pneumophila (L.p.) is a gram-negative, intracellular pathogen and a common cause of severe community-acquired pneumonia. In humans, L.p. replicates primarily within alveolar macrophages. It manipulates vital host cell functions such as vesicle trafficking and gene expression by the secretion of over 300 effector proteins into the host cell cytosol. Thus, L.p. modifies its host cell to promote its own replication. An unbiased and global analysis of the molecular changes and biological processes that are associated with bacterial infections of human cells can provide new insights into host-pathogen interactions. Therefore, one goal of this study was to characterize expression changes of different RNA species in response to infection with L.p. in human primary blood-derived macrophages (BDMs) or differentiated THP-1 cells. This work is structured into two parts: (1) a functional study on how miRNA manipulations can alter L.p. replication in macrophages and (2) an in depth analysis of transcriptomic events in host and pathogen during infection. (1) In the last few decades, miRNAs have been established as critical modulators of immune function. Therefore, one aim of this study was to identify the miRNA profile of L.p.-infected macrophages and to determine the functional impact of a miRNA manipulation on L.p. replication. BDMs of healthy donors were infected with L.p. strain Corby. Small RNA sequencing revealed the miRNA profile in BDMs following L.p. infection. An upregulation of miR-146a and miR-155, as well as downregulation of miR-221 and miR-125b was validated by qPCR in macrophages. miRNA regulation in response to infection seems to be due to transcriptional regulation of miRNA promoters, since the histone acetylation levels at the promoter and the pri-miR expression correlated with the miRNA expression upon L.p.- infection. Overexpression and knock down experiments of miR-125b, miR-221 and miR-579 in combination were performed for functional characterization and showed an influence of all three miRNAs on bacterial replication. A SILAC approach revealed the protein MX1 as downregulated following simultaneous overexpression of all three miRNAs. MX1 is an interferon-induced GTP-binding protein important for antiviral defence. As shown by validation experiments, MX1 knockdown in macrophages led to an increased replication of L.p., as seen following overexpression of the miRNAs. Since in silico analysis predicted no binding sites for either miRNA in the 3’UTR of MX1, Ingenuity pathway analysis was performed to find the linking molecules. DDX58 (RIG-I), a sensor for cytosolic RNA, was validated as a target for miR-221, while the tumour suppressor TP53 was shown to be targeted by miR-125b via luciferase reporter assays. An siRNA-mediated knockdown of both, TP53 and DDX58, respectively, led to an enhanced replication of L.p. in macrophages. Thus, DDX58 and TP53 were validated as linking molecules between the three miRNAs and MX1. Additionally, the aforementioned SILAC approach revealed a downregulation of LGALS8 which was later validated as a target of miR-579. LGALS8 is a cytosolic lectin which binds carbohydrates and localizes to damaged vesicles. Knockdown of LGALS8 enhanced intracellular replication in macrophages. Thus, MX1 and LGALS8 were identified as targets of the three miRNAs (miR-125b, miR-221, miR-579) and to be responsible for the restriction of L.p. replication within human macrophages. (2) The transcriptional profile of L.p. during the course of infection in human macrophages was next to be established. Dual RNA-Sequencing was performed to determine the regulation of coding and non-coding RNA species during the course of infection of both, host and pathogen, simultaneously. After adaptation and optimization of existing protocols, macrophages were infected using a GFP-expressing L.p. strain Corby. To separate infected cells (gfp+) from the non-invaded bystander cells (gfp-), flow cytometry sorting was performed. Furthermore, Pam3CSK4 was used to generate TLR2-activated cells. RNA from all different samples, and also RNA from cultivated Legionella, was sequenced. Differential gene expression analysis was performed using DESeq2 resulting in 4,144 differentially expressed human genes (across multiple conditions) and 2,707 differentially expressed Legionella genes (across two time points). The DESeq analysis of the separated RNA fractions from host cells revealed differentially expressed mRNAs (3,504), lncRNAs (495), and miRNAs (145). 1,128 differentially expressed genes were exclusively significantly regulated in invaded cells (gfp+ at 8 and 16 h). Some of these were validated via qPCR including BCL10, SOD1, IRS1, CYR61, ATG5, RND3 and JUN. In addition, the simultaneous upregulation of the genes ZFAND2A and HSPA1 in the bystander and in Legionella-invaded cells was validated. The analysis of the bacterial mRNAs revealed a switch of gene usage, i.e. inverse regulation at 8 and 16 h post infection. This switch included genes which are involved in iron metabolism, stress response, glycolysis and lipid biosynthesis. Hence, differentially expressed genes within different growth phases of the infection cycle were identified. This dataset is the first of its kind to cover a respiratory pathogen. The dual RNA-Sequencing performed in this study provides data to encapsulate the RNA landscape of coding and non-coding RNAs in pathogen and host. In summary, the results have deepened our insight into the infection process and the molecular interaction of L.p. and its host cells and will help to understand the complex interplay between host and pathogen by allowing for the in silico re-construction of an RNA interaction network. Furthermore, the present study will help to establish potential new candidates for diagnosis and therapy

Investigation of zebrafish gills as a model to study respiratory immunology

Respiratory viral infections are routinely studied with animal models or in vitro culture which mimic but do not fully recapitulate elements of human immune responses or pathology. Studying events at the respiratory mucosa where infection is established is crucial to further our understanding of immune responses to these infections. In this thesis I evaluated the zebrafish gills as a novel in vivo model to study respiratory immunity. Using transgenic zebrafish, flow cytometry and several microscopy techniques I characterized immune cell composition and function in this tissue. In addition, I assessed the stimulatory effects of pathogen mimetics and derivatives on immune response pathways using RNA analysis. The stimulatory and pathological effects of live viral challenges were also assessed. These experiments highlighted the abundance and diversity of innate and adaptive immune cells in the gills from early to adult developmental stages. Some of these immune cells were capable of antigen-uptake and formed dynamic cell-cell interactions. Aggregates of T and B cells were identified as interbranchial lymphoid tissue which had some similarities but also some architectural differences to mammalian mucosal-associated lymphoid tissue. Different responses were stimulated by resiquimod and lipopolysaccharide highlighting these pathogen-related compounds as tools to interrogate immune pathways in the gills. On the other hand, zebrafish had limited responses to human respiratory or fish-endemic viruses and were resistant to pathological infection. These results highlight zebrafish gills as a useful model to investigate several aspects of immune cell function and inflammation in a respiratory mucosal in vivo setting. Further investigation with pathogens is needed to expand the use of this model for infection studies.Open Acces

Systematic analysis of small RNA function in respiratory virus infection

Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are among the most common causes of respiratory infections worldwide, causing morbidity and mortality especially in young children but also elderly or immunocompromised adults. Infections result in hundreds of thousands of hospitalizations each year, leading also to a significant global health and economic burden. RSV alone infects nearly all children by the age of 2 years and is estimated to cause a higher disease burden than influenza in the adult population. Despite intensive research, there are still no licenced vaccines or effective treatments against RSV and PIV infections. Most antiviral approaches to date directly target the virus, although many host factors are involved in the viral replication cycle. MicroRNAs (miRNAs) are a class of small regulatory RNAs that canonically supress gene expression by binding to the 3’ untranslated region (3’ UTR) of messenger RNAs (mRNAs). Several miRNAs have been reported to be altered upon viral infection and these alterations can suppress or boost host immune responses and the viral infection progress, depending on the targets of the miRNAs. However, there is no systematic analysis of miRNA and target regulation in respiratory virus infection. Previous functional screening from our group using miRNA mimics and inhibitors demonstrated that some miRNAs including miR-744 and miR-28 have antiviral properties against a wide range of viruses. However, the targets of these miRNAs and their mechanisms of action remain largely unknown. This thesis focuses on the global characterisation of host miRNA regulation in RSV and PIV-3 infections in the A549 epithelial cell line and then further investigates the targets of key de-regulated or antiviral miRNAs in RSV infections. Dynamic changes in miRNA expression over the time course of 96 h of RSV and PIV-3 infections were determined by small RNA sequencing. In general, RSV and PIV-3 induced similar de-regulation of miRNA levels, with miR-149 and miR-744 being down-regulated and the miR-34/miR-449 cluster being upregulated. Other miRNAs that we previously identified as antiviral (such as miR-28) were not differentially expressed. In addition, several viral small RNAs (vsRNAs) with a length of 22-30 nt were identified in the small RNA sequencing data for both RSV and PIV-3. The RSV vsRNAs were found to be present not only inside the cell but also in cell culture supernatant at early time points of infection. Small RNA sequencing from RSV-infected patients confirmed their presence in bronchial alveolar lavage fluid. Inhibition of one of these RNAs (vsRNA L-1) inhibited RSV infection and replication, making this RNA an interesting candidate for future therapeutic approaches. To investigate the mechanism of action of deregulated and antiviral miRNAs in RSV infection, miRNA targets were identified by immunoprecipitation of the RNA-induced silencing complex (RISC), followed by ligation of the miRNA to its target and sequencing of these chimeric RNAs. After initial optimisation, two protocols were tested side by side to identify optimal conditions for the A549 cell line. A variation of the CLEAR-CLIP (covalent ligation of endogenous Argonaute-bound RNAs– Crosslinking and immunoprecipitation) protocol resulted in 3-6% of miRNA-target interactions. Apart from canonical interactions with 3’ UTRs, high confidence targets were identified in coding sequences (CDS) and regulatory regions using a new bioinformatic approach developed by our laboratory. In general, miRNA expression correlated with the number of target counts. However, for a subset of miRNAs that were highly abundant, no high-confidence targets could be identified, including the miR-449 family. These data suggest that additional mechanisms, such as RISC association, regulate targeting of these miRNAs. In addition to the identification of host targets, this analysis also identified bindings sites in the viral genome for host miRNAs miR-26 and miR-27, which could represent a viral mechanism to suppress these host miRNAs and up-regulate host gene expression. High confidence targets were analysed in detail for a subset of lead candidate miRNAs to identify their mechanisms of action. Target network analysis suggested miR-26 and miR-27 as regulators of host genes important for viral processes. Too few targets were found to perform target network analysis for miR-28 or miR-744, but initial results confirm the Signal Peptidase Complex Subunit 3 (SPCS3) 3’ UTR as a target for miR-28. This gene was reported to be essential in viral infections and could explain the antiviral effect of miR-28 mimics. In summary, this thesis provides a global overview of miRNA regulation during the time course of respiratory virus infections and provides insight into new target interaction of de-regulated or antiviral miRNAs during RSV infection. Expanding our knowledge of miRNA-mediated gene regulations in the context of infection will contribute to a better understanding of pathways that could be targeted in new therapeutic strategies

Efficacy and safety of metabolic interventions for the treatment of severe COVID-19: in vitro, observational, and non-randomized open-label interventional study

Background: Viral infection is associated with a significant rewire of the host metabolic pathways, presenting attractive metabolic targets for intervention. Methods: We chart the metabolic response of lung epithelial cells to SARS-CoV-2 infection in primary cultures and COVID-19 patient samples and perform in vitro metabolism-focused drug screen on primary lung epithelial cells infected with different strains of the virus. We perform observational analysis of Israeli patients hospitalized due to COVID-19 and comparative epidemiological analysis from cohorts in Italy and the Veteran's Health Administration in the United States. In addition, we perform a prospective non-randomized interventional open-label study in which 15 patients hospitalized with severe COVID-19 were given 145 mg/day of nanocrystallized fenofibrate added to the standard of care. Results: SARS-CoV-2 infection produced transcriptional changes associated with increased glycolysis and lipid accumulation. Metabolism-focused drug screen showed that fenofibrate reversed lipid accumulation and blocked SARS-CoV-2 replication through a PPARα-dependent mechanism in both alpha and delta variants. Analysis of 3233 Israeli patients hospitalized due to COVID-19 supported in vitro findings. Patients taking fibrates showed significantly lower markers of immunoinflammation and faster recovery. Additional corroboration was received by comparative epidemiological analysis from cohorts in Europe and the United States. A subsequent prospective non-randomized interventional open-label study was carried out on 15 patients hospitalized with severe COVID-19. The patients were treated with 145 mg/day of nanocrystallized fenofibrate in addition to standard-of-care. Patients receiving fenofibrate demonstrated a rapid reduction in inflammation and a significantly faster recovery compared to patients admitted during the same period. Conclusions: Taken together, our data suggest that pharmacological modulation of PPARα should be strongly considered as a potential therapeutic approach for SARS-CoV-2 infection and emphasizes the need to complete the study of fenofibrate in large randomized controlled clinical trials. Funding: Funding was provided by European Research Council Consolidator Grants OCLD (project no. 681870) and generous gifts from the Nikoh Foundation and the Sam and Rina Frankel Foundation (YN). The interventional study was supported by Abbott (project FENOC0003). Clinical trial number: NCT04661930