RNA-Seq of Influenza A virus infected THP-1 cells at different timepoint
Ontology highlight
ABSTRACT: THP-1 cells were infected by IAV virus strain X31 with a MOI=10, and cells were collected at 3 timepoints:0 hr, 24 hr and 48 hr post-infection. Each timepoint has 3 biological replicates.
Project description:GFP-tagged DARPin F10 and CTR are expressed in A549 cells, then GFP positive cells are sorted by GFP positive with FACS. Cells are pelleted and total RNA is extracted. The aim is to check DARPin F10 effects on gene expression.
Project description:De novo synthesized protein DARPin F10 can target HDAC6 ZnF domain, and inhibits Influenza A virus and ZIKA virus infection. Our data shows it interferes with the virus uncoating step. To exclude the possibility that F10 upregulates interferon pathways, we checked the transcription of the cell
Project description:Influenza A Virus (IAV) is a recurring respiratory virus with antiviral therapies of limited use. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses with three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we mapped 332 IAV-human protein-protein interactions and identified 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza revealed several genes, including the structural scaffold protein AHNAK, with predicted loss-of-function variants that were also identified in our proteomic analyses. Of our identified host factors, 54 significantly altered IAV infection upon siRNA knockdown, and two factors, COPB1 and AHNAK, were also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppressed IAV replication, with three targeting ATP6V1A, CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT. This project includes the global proteomic data (abundance and phosphorylation), the AP-MS data has been submitted separately as its own dataset and has its own dataset identifier.
Project description:Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes. Here, we demonstrate that the viral hemagglutinin (HA) mediates bacterial OM by inducing a pro-inflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings show that it is the inflammatory response that mediates pneumococcal replication; not viral suppression of the immune system or epithelial damage. This study provide the first evidence that HA induced inflammation drives pneumococcal replication in the middle ear cavity, which has important consequences to the treatment of pneumococcal OM. Five-day old C57BL/6 mice were colonised intranasally (i.n.) with 2M-CM-^W103 colony forming units (CFU) of S. pneumoniae EF3030Lux in 3 M-BM-5Ls of PBS. Alternatively, mice were mock-infected with an equivalent volume of phosphate buffered saline (PBS). At 14-days of age, infant mice were infected i.n. with 20 plaque forming units (PFU) (PR8/34, Cambridge/34 and WSN/33) or 102.5 PFU (all other virus strains) of egg-grown IAV in 3 M-BM-5Ls of PBS. Viral doses were selected to ensure a reproducible infection with minimum morbidity and no mortality. Six days post-IAV infection, mice were euthanised and organs were collected for analysis. Six independent biological replicates (where both ears from one mouse were pooled to create one sample) were used for each condition and analyzed using the NimbleGen platform (12M-CM-^W135K Mouse Gene Expression Arrays, Roche Nimblegen, USA). Indirect labeling, hybridization and washing was performed according to the manufacturerM-bM-^@M-^Ys instructions. Array images were acquired with a NimbleGen MS200 scanner, and images were processed with NimbleScan software using the RMA algorithm. Data was processed using Arraystar (DNASTAR, USA) with default settings as described in the manual. Differential expression tests were performed with a moderated T-test implemented in Arraystar, followed by FDR correction of the P values (Q-values) according to the method of Storey and Tibshirani
Project description:Influenza A Virus (IAV) is a recurring respiratory virus with antiviral therapies of limited use. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses with three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we mapped 332 IAV-human protein-protein interactions and identified 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza revealed several genes, including the structural scaffold protein AHNAK, with predicted loss-of-function variants that were also identified in our proteomic analyses. Of our identified host factors, 54 significantly altered IAV infection upon siRNA knockdown, and two factors, COPB1 and AHNAK, were also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppressed IAV replication, with three targeting ATP6V1A, CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT. This project includes the AP-MS data; all global proteomic data (abundance and phosphorylation) has been submitted separately as its own dataset and has its own dataset identifier.
Project description:Severe bacterial (pneumococcal) infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Disruption of lung tissue integrity during influenza participates in bacterial pulmonary colonization and dissemination out of the lungs. Interleukin (IL)-22 has gained considerable interest in anti-inflammatory and anti-infection immunotherapy over the last decade. In the current study, we investigated the effect of exogenous IL-22 delivery on the outcome of bacterial superinfection post-influenza. Our data show that exogenous treatment of influenza-infected mice with recombinant IL-22 reduces bacterial dissemination out of the lungs but is without effect on pulmonary bacterial burden. We describe an IL-22 specific gene signature in the lung tissue of IAV-infected (and naïve) mice that might explain the observed effects. Indeed, exogenous IL-22 modulates gene expression profile in a way suggesting a reinforcement of tissue integrity. Our results open the way to alternative approaches for limiting post-influenza bacterial superinfection, particularly systemic bacterial invasion.
Project description:Influenza A virus (IAV) is a zoonotic pathogen causing respiratory infections in humans and other mammalian species. Besides the potential to cause pandemics, seasonal IAV causes high medical and economical burden due to 3 to 5 million cases of severe respiratory illness and up to 500,000 deaths every year. Increasing resistance against clinically used anti influenza drugs and an insufficient vaccine protection urge the development of new antiviral strategies to counteract this constant threat to global health. One new approach focuses on cellular factors involved in the IAV life cycle as potential drug targets. Particularly promising are kinases and their target proteins, as kinase inhibitors comprise up to 30% of drug discovery programs in the pharmaceutical industry. In this project, we aim to find suitable candidates for the development of host factor targeted antivirals by using state-of-the-art quantitative phosphoproteomics to reveal the unique phosphoproteome dynamics that occur in the host cell within minutes of IAV infection and enable entry of the virus into its host cell. We identified 3,920 host proteins phosphorylated by infection with avian and seasonal human IAV strains. Among them are known entry factors such as the human epidermal growth factor receptor (EGFR) and members of the phosphoinositid-3-kinase (PI3K) pathway, which validate our approach.
Project description:Influenza A viruses (IAVs) are respiratory pathogens that represent a global concern due to their ability to cause seasonal epidemics and sporadic pandemics in the human population. Thus, a comprehensive understanding of IAV biology is essential in order to design effective antivirals. It is well established that entry of most IAVs begins through binding to cell surface sialic acid, however the identity of receptors that mediate virus internalization remains unclear. Here, we performed TurboID-based proximity labelling centered on epsin 1, a protein required for clathrin-mediated endocytosis of IAV, to uncover internalization receptors. By performing affinity-based purification of biotinylated proteins coupled to mass spectrometry, we identified 34 proteins proximal to epsin 1 during IAV infection. Therein we found 11 high-confidence STRING interactors for epsin 1 and 3 putative pro-viral host factors for IAV infection, validating our approach.
Project description:This study investigates the role of store-operated calcium entry (SOCE) in T cell-mediated immune responses to pulmonary influenza A virus (IAV) infection and allergic airway inflammation after immunization by house dust mite (HDM) allergens. We conducted a comparative gene expression analysis of antigen-specific CD4+ T cells from wildtype (WT) and Orai1fl/fl Cd4Cre mice that were adoptively transferred to TCRalpha knockout mice followed by sensitization / challenge with HDM or infection with influenza A virus (IAV) strain x31. Donor CD4+ T cells were isolated from the lungs of host mice at days 9 (IAV) and 14 (HDM), RNA was isolated and processed for bulk RNA sequencing.