MicroRNAs affect GPCR and Ion channel genes needed for influenza replication.
Ontology highlight
ABSTRACT:
SUBMITTER: Orr-Burks N
PROVIDER: S-EPMC8742985 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Influenza virus causes epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses. Influenza viruses require host genes to replicate. RNA interference (RNAi) screens can identify host genes coopted by influenza virus for replication. Targeting these proinfluenza genes can provide therapeutic strategies to reduce virus replication. Nineteen proinfluenza G-protein-coupled receptor (GPCR) and 13 proinfluenza ion channel genes were identified in human lung (A549) cells by use of small interfering RNAs (siRNAs). These proinfluenza genes were authenticated by testing influenza virus A/WSN/33-, A/CA/04/09-, and B/Yamagata/16/1988-infected A549 cells, resulting in the validation of 16 proinfluenza GPCR and 5 proinfluenza ion channel genes. These findings showed that several GPCR and ion channel genes are needed for the production of infectious influenza virus. These data provide potential targets for the development of host-directed therapeutic strategies to impede the influenza virus productive cycle so as to limit infection.IMPORTANCE Influenza epidemics result in morbidity and mortality each year. Vaccines are the most effective preventive measure but require annual reformulation, since a mismatch of vaccine strains can result in vaccine failure. Antiviral measures are desirable particularly when vaccines fail. In this study, we used RNAi screening to identify several GPCR and ion channel genes needed for influenza virus replication. Understanding the host genes usurped by influenza virus during viral replication can help identify host genes that can be targeted for drug repurposing or for the development of antiviral drugs. The targeting of host genes is refractory to drug resistance generated by viral mutations, as well as providing a platform for the development of broad-spectrum antiviral drugs.
| S-EPMC8104092 | biostudies-literature
Project description:BackgroundAlveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60) are frequently used to model macrophage function.MethodsThe availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR) and ion channel expression in alveolar macrophages and their widely used surrogates.ResultsThe complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates.ConclusionsThe data described in this report provides insight into the appropriate choice of cell models for investigating macrophage biology and highlights the importance of confirming experimental data in primary alveolar macrophages.
| S-EPMC3542584 | biostudies-literature
Project description:Human protein kinases (HPKs) have profound effects on cellular responses. To better understand the role of HPKs and the signaling networks that influence influenza virus replication, a small interfering RNA (siRNA) screen of 720 HPKs was performed. From the screen, 17 HPKs (NPR2, MAP3K1, DYRK3, EPHA6, TPK1, PDK2, EXOSC10, NEK8, PLK4, SGK3, NEK3, PANK4, ITPKB, CDC2L5 (CDK13), CALM2, PKN3, and HK2) were validated as essential for A/WSN/33 influenza virus replication, and 6 HPKs (CDK13, HK2, NEK8, PANK4, PLK4 and SGK3) were identified as vital for both A/WSN/33 and A/New Caledonia/20/99 influenza virus replication. These HPKs were found to affect multiple host pathways and regulated by miRNAs induced during infection. Using a panel of miRNA agonists and antagonists, miR-149* was found to regulate NEK8 expression, miR-548d-3p was found to regulate MAPK1 transcript expression, and miRs -1228 and -138 to regulate CDK13 expression. Up-regulation of miR-34c induced PLK4 transcript and protein expression and enhanced influenza virus replication, while miR-34c inhibition reduced viral replication. These findings identify HPKs important for influenza viral replication and show the miRNAs that govern their expression.
| S-EPMC3689682 | biostudies-literature
Project description:Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.
| S-EPMC5934295 | biostudies-literature
Project description:Ion channels serve many cellular functions including ion homeostasis, volume regulation, signaling, nutrient acquisition, and developmental progression. Although the complex life cycles of malaria parasites necessitate ion and solute flux across membranes, the whole-genome sequencing of the human pathogen Plasmodium falciparum revealed remarkably few orthologs of known ion channel genes. Contrasting with this, biochemical studies have implicated the channel-mediated flux of ions and nutritive solutes across several membranes in infected erythrocytes. Here, I review advances in the cellular and molecular biology of ion channels in malaria parasites. These studies have implicated novel parasite genes in the formation of at least two ion channels, with additional ion channels likely present in various membranes and parasite stages. Computational approaches that rely on homology to known channel genes from higher organisms will not be very helpful in identifying the molecular determinants of these activities. Given their unusual properties, novel molecular and structural features, and essential roles in pathogen survival and development, parasite channels should be promising targets for therapy development.
| S-EPMC10970509 | biostudies-literature
Project description:Influenza virus, a negative-stranded RNA virus that causes severe illness in humans and animals, stimulates the inflammasome through the Nod-like receptor NLRP3. However, the mechanism by which influenza virus activates the NLRP3 inflammasome is unknown. Here we show that the influenza virus M2 protein, a proton-selective ion channel important in viral pathogenesis, stimulates the NLRP3 inflammasome pathway. M2 channel activity was required for the activation of inflammasomes by influenza and was sufficient to activate inflammasomes in primed macrophages and dendritic cells. M2-induced activation of inflammasomes required its localization to the Golgi apparatus and was dependent on the pH gradient. Our results show a mechanism by which influenza virus infection activates inflammasomes and identify the sensing of disturbances in intracellular ionic concentrations as a previously unknown pathogen-recognition pathway.
| S-EPMC2857582 | biostudies-literature
Project description:MicroRNAs (miRNAs) are a class of noncoding RNAs of lengths ranging from 18 to 23 nucleotides (nt) that play critical roles in a wide variety of biological processes. There is a growing amount of evidence that miRNAs play critical roles in intricate host-pathogen interaction networks, but the involvement of miRNAs during influenza viral infection is unknown. To determine whether the cellular miRNAs play an important role in H1N1 influenza A viral infections, 3' untranslated region (UTR) reporter analysis was used to identify putative miRNA targets in the influenza virus genome, and virus proliferation analysis was used to detect the effect of the screened miRNAs on the replication of H1N1 influenza A virus (A/WSN/33) in MDCK cells. The results showed that miRNA 323 (miR-323), miR-491, and miR-654 inhibit replication of the H1N1 influenza A virus through binding to the PB1 gene. Moreover mutational analysis of the predicted miRNA binding sites showed that the three miRNAs bind to the same conserved region of the PB1 gene. Intriguingly, despite the fact that the miRNAs and PB1 mRNA binding sequences are not a perfect match, the miRNAs downregulate PB1 expression through mRNA degradation instead of translation repression. This is the first demonstration that cellular miRNAs regulate influenza viral replication by degradation of the viral gene. Our findings support the notion that any miRNA has antiviral potential, independent of its cellular function, and that the cellular miRNAs play an important role in the host, defending against virus infection.
| S-EPMC2919005 | biostudies-literature
Project description:Endogenous ions play important roles in the function and pharmacology of G-protein coupled receptors (GPCRs). Historically the evidence for ionic modulation of GPCR function dates to 1973 with studies of opioid receptors, where it was demonstrated that physiologic concentrations of sodium allosterically attenuated agonist binding. This Na+-selective effect was distinct from effects of other monovalent and divalent cations, with the latter usually counteracting sodium's negative allosteric modulation of binding. Since then, numerous studies documenting the effects of mono- and divalent ions on GPCR function have been published. While ions can act selectively and nonselectively at many sites in different receptors, the discovery of the conserved sodium ion site in class A GPCR structures in 2012 revealed the unique nature of Na+ site, which has emerged as a near-universal site for allosteric modulation of class A GPCR structure and function. In this review, we synthesize and highlight recent advances in the functional, biophysical, and structural characterization of ions bound to GPCRs. Taken together, these findings provide a molecular understanding of the unique roles of Na+ and other ions as GPCR allosteric modulators. We will also discuss how this knowledge can be applied to the redesign of receptors and ligand probes for desired functional and pharmacological profiles. SIGNIFICANCE STATEMENT: The function and pharmacology of GPCRs strongly depend on the presence of mono and divalent ions in experimental assays and in living organisms. Recent insights into the molecular mechanism of this ion-dependent allosterism from structural, biophysical, biochemical, and computational studies provide quantitative understandings of the pharmacological effects of drugs in vitro and in vivo and open new avenues for the rational design of chemical probes and drug candidates with improved properties.
| S-EPMC6782022 | biostudies-literature
Project description:BACKGROUND:PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. METHODOLOGY/PRINCIPAL FINDINGS:Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAV(PR8)), from A/Brevig Mission/1/1918(H1N1) (IAV(SF2)) or the H5N1 consensus sequence (IAV(BF2)) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. CONCLUSION/SIGNIFICANCE:The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development.
| S-EPMC2886074 | biostudies-literature
Project description:BACKGROUND:A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a "mixing vessel" for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. RESULTS:To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2-cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2-cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2-cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2-cMyc RNA or a buffer were -152 and -200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were -141 and -146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were -21 and -22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential. CONCLUSION:The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.
| S-EPMC6013169 | biostudies-literature