ORF3a of SARS-CoV-2 modulates PI3K/AKT signaling in human lung epithelial cells via hsa-miR-155-5p.

SARS-CoV-2 infection results in cytokine burst, leading to proinflammatory responses in lungs of COVID-19 patients. SARS-CoV-2 ORF3a triggers the generation of proinflammatory cytokines. However, the underlying mechanism of dysregulation of proinflammatory responses is not well understood. We studied the role of microRNA in the generation of proinflammatory responses as a bystander effect of SARS-CoV-2 ORF3a in human lung epithelial cells. We observed upregulation of hsa-miR-155-5p in SARS-CoV-2 ORF3a transfected human lung epithelial cells, which led to the reduced expression of SHIP1. This resulted in phosphorylation of AKT and NF-κB, which further led to the increased expression of the proinflammatory cytokines IL-6 and TNF-α. Additionally, overexpression and knockdown studies of hsa-miR-155-5p were performed to confirm the role of hsa-miR-155-5p in the regulation of the SHIP1. We demonstrated that hsa-miR-155-5p modulates the proinflammatory response by activating the PI3K/AKT pathway through the inhibition of SHIP1 in SARS-CoV-2 ORF3a transfected human lung epithelial cells.

Chandipura virus changes cellular miRNome in human microglial cells.

Chandipura virus (CHPV) is a neurotropic virus, known to cause encephalitis in humans. The microRNAs (miRNA/miR) play an important role in the pathogenesis of viral infection. The present study is focused on the role of miRNAs during CHPV (strain 1653514) infection in human microglial cells. The deep sequencing of CHPV-infected human microglial cells identified a total of 12 differentially expressed miRNA (DEMs). To elucidate the role of DEMs, the target gene prediction, Gene Ontology term (GO Term), pathway enrichment analysis, and miRNA-messenger RNA (mRNA) interaction network analysis was performed. The GO terms and pathway enrichment analysis provided 146 enriched genes; which were involved in interferon response, cytokine and chemokine signaling. Further, the WGCNA (weighted gene coexpression network analysis) of the enriched genes were discretely categorized into three modules (blue, brown, and turquoise). The hub genes in the blue module may correlate to CHPV induced neuroinflammation. Altogether, the miRNA-mRNA interaction network and WGCNA study revealed the following pairs, hsa-miR-542-3p and FAF1, hsa-miR-92a-1-5p and MYD88, and hsa-miR-3187-3p and TNFRSF21, which may contribute to neuroinflammation during CHPV infection in human microglial cells.

Chikungunya virus modulates the miRNA expression patterns in human synovial fibroblasts.

Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes. CHIKV infection leads to polyarthritis and polyarthralgia among patients. The synovial fibroblasts are the primary target for CHIKV. The microRNAs (miRNAs) are the small endogenous noncoding RNAs which posttranscriptionally regulate the expression of genes by binding to their target messenger RNAs (mRNAs) through their 3'-untranslated regions. The miRNAs are the key regulators for various pathological processes including viral infection, cancer, cardiovascular disease, and neurodegeneration. This study was designed to dissect out the roles of miRNAs during CHIKV (Ross Strain E1: A226V) infection in primary human synovial fibroblasts. The miRNA microarray profiling was performed on the primary human synovial fibroblasts infected by CHIKV. The gene target prediction analysis, enrichment, and network analysis were performed by various bioinformatics analyses. The subset of 26 differentially expressed microRNAs (DEMs) were identified through microarray profiling and were further screened for gene predictions, Gene Ontology, pathway enrichment, and miRNA-mRNA network using various bioinformatics tools. The bioinformatics analysis indicates the role of DEMs by suppressing the immune response which may contribute to CHIKV persistence in human primary synovial fibroblasts. Our study provides the plausible roles of DEMs, miRNAs, and mRNA interactions and pathways involved in the molecular pathogenesis of CHIKV.

Modulation of Type-I Interferon Response by hsa-miR-374b-5p During Japanese Encephalitis Virus Infection in Human Microglial Cells.

Japanese Encephalitis virus (JEV) is a neurotropic ssRNA virus, belonging to the Flaviviridae family. JEV is one of the leading causes of the viral encephalitis in Southeast-Asian countries. JEV primarily infects neurons however, the microglial activation has been reported to further enhance the neuroinflammation and promote neuronal death. The PI3K/AKT pathway has been reported to play an important role in type-I interferon response via IRF3. Phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/AKT pathway, participates in microglial polarization and neuroinflammation. The microRNAs are small non-coding endogenously expressed RNAs, which regulate the gene expression by binding at 3' UTR of target gene. The human microglial cells were infected with JEV (JaOArS982 strain) and up-regulation of microRNA; hsa-miR-374b-5p was confirmed by qRT-PCR. The genes in PI3K/AKT pathway, over-expression and knock-down studies of hsa-miR-374b-5p with and without JEV infection were analyzed through immuno blotting. The regulatory role of hsa-miR-374b-5p on the expression of type-I interferon was determined by luciferase assays. JEV infection modulated the expression of hsa-miR-374b-5p and PI3K/AKT pathway via PTEN. The over-expression of hsa-miR-374b-5p suppressed the PTEN while up-regulated the AKT and IRF3 proteins, whereas, the knockdown rescued the PTEN expression and suppressed the AKT and IRF3 proteins. The modulation of hsa-miR-374b-5p regulated the type-I interferon response during JEV infection. In present study, we have shown the modulation of PTEN by hsa-miR-374b-5p, which regulated the PI3K/AKT/IRF3 axis in JEV infected microglial cells.

Exploitation of microRNAs by Japanese Encephalitis virus in human microglial cells.

JEV infection in CNS leads to the JE neuroinflammation. Children and old age individual have been reported to be more prone to JEV infection. MicroRNAs are endogenous, small non-coding RNAs, which regulate the gene expression. These are ∼22 nucleotide long, conserved RNA sequence that binds at the 3'UTR of a target mRNA and regulate the post-transcriptional gene expression. The role of microRNAs has been reported in several diseases like cancer, viral infection, neuro-degeneration, diabetes etc. In the present study, the human microglial cells were infected with JEV (JaOArS982). The control and infected samples were subject to microarray profiling for microRNA expression. The microarray profile yielded differentially expressed microRNAs from JEV infected samples. The microRNA gene targets, gene ontology, annotations, and pathways were identified through various bioinformatics tools. Additionally, the pathways were mostly found common to "ubiquitin mediated proteolysis," "cytokine signaling," "maintenance of barrier function/cell junctions," JAK/STAT pathway" "Toll-like receptor signaling," "Wnt-signaling," "adhesion molecules," "apoptosis," "endocytosis," "vesicle mediated transport" etc.

HIV-1 Tat C modulates NOX2 and NOX4 expressions through miR-17 in a human microglial cell line.

HIV-1 invades CNS in the early course of infection, which can lead to the cascade of neuroinflammation. NADPH oxidases (NOXs) are the major producers of reactive oxygen species (ROS), which play important roles during pathogenic insults. The molecular mechanism of ROS generation via microRNA-mediated pathway in human microglial cells in response to HIV-1 Tat protein has been demonstrated in this study. Over-expression and knockdown of microRNAs, luciferase reporter assay, and site-directed mutagenesis are main molecular techniques used in this study. A significant reduction in miR-17 levels and increased NOX2, NOX4 expression levels along with ROS production were observed in human microglial cells upon HIV-1 Tat C exposure. The validation of NOX2 and NOX4 as direct targets of miR-17 was done by luciferase reporter assay. The over-expression and knockdown of miR-17 in human microglial cells showed the direct role of miR-17 in regulation of NOX2, NOX4 expression and intracellular ROS generation. We demonstrated the regulatory role of cellular miR-17 in ROS generation through over-expression and knockdown of miR-17 in human microglial cells exposed to HIV-1 Tat C protein. Activated microglial cells mediated neuroinflammatory events are observed in HIV-associated neurological disorders. The reduction in miR-17 levels was observed in microglial cells exposed to HIV-1 Tat C protein. miR-17 regulated the expression of NOX2 and NOX4, which in turn regulated the reactive oxygen species (ROS) production in microglial cells. Increased ROS production led to the activation of microglial cells and increased cytokine production. This study thus demonstrated a novel miR-17-mediated regulatory pathway of ROS production in microglial cells. HMC3 = human microglia clone 3 cell lines.

Neurovirology: neurotropic viruses and the brain. Interview with Sunit K Singh by Hannah Branch.

Sunit K Singh speaks to Hannah Branch, Commissioning Editor Sunit K Singh obtained a Bachelor's degree from GB Pant University of Agriculture and Technology (India) followed by his Master's degree from the Central Institute of Fisheries Education (India). Upon the completion of his Master's degree, Singh moved to the University of Wuerzburg (Germany) to complete his PhD degree in the field of Molecular Infection Biology. Subsequently, he undertook his postdoctoral training at Yale University School of Medicine (CT, USA) and at the University of California Davis Medical Center (CA, USA). Singh has also been visiting faculty at the Albert Einstein College of Medicine (NY, USA), Department of Pathology and Immunology, University of Geneva (Switzerland) and the Institute of Parasitology (Czech Republic). At present, he is a permanent member of faculty in the Section of Infectious Diseases and Immunobiology Centre for Cellular and Molecular Biology (CCMB; India) and is leading a research group in the area of neurovirology there. Singh has been an author of a number of papers published in peer-reviewed journals, and has been honored with several awards, including the Skinner Memorial Award, Young Scientist Award, Travel Grant Award and the NIH-Fogarty Fellowship. In addition, he is a valued editorial board member of several journals, including Future Microbiology.

Progress towards therapeutic application of RNA interference for HIV infection.

HIV-1 infection is the cause of acquired immune deficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) has been successful in reducing the rate of progression to AIDS, but a cure has not yet been achieved. New tools are required to delay progression of infection or to block the replication cycle of HIV. RNA interference (RNAi) has the potential to work as a powerful tool against HIV infection. The mode of action of small interfering RNAs (siRNAs) against their target genes is through sequence complementarity, which in turn results in target degradation. siRNAs are showing enormous potential to be used as a therapeutic tool in various diseases; however, this technology still requires refinement before its full potential can be utilized for the development of HIV therapies.

MicroRNAs--micro in size but macro in function.

MicroRNAs (miRNAs) are endogenous small RNAs that can regulate target mRNAs by binding to their 3'-UTRs. A single miRNA can regulate many mRNA targets, and several miRNAs can regulate a single mRNA. These have been reported to be involved in a variety of functions, including developmental transitions, neuronal patterning, apoptosis, adipogenesis metabolism and hematopoiesis in different organisms. Many oncogenes and tumor suppressor genes are regulated by miRNAs. Studies conducted in the past few years have demonstrated the possible association between miRNAs and several human malignancies and infectious diseases. In this article, we have focused on the mechanism of miRNA biogenesis and the role of miRNAs in human health and disease.

Inflammation in white matter: clinical and pathophysiological aspects.

While the central nervous system (CNS) is generally thought of as an immunopriviledged site, immune-mediated CNS white matter damage can occur in both the perinatal period and in adults, and can result in severe and persistent neurological deficits. Periventricular leukomalacia (PVL) is an inflammatory white matter disease of premature infants that frequently results in cerebral palsy (CP). Clinical and experimental studies show that both hypoxic/ischemic and innate immune mechanisms contribute to the destruction of immature oligodendroglia and of axons in the deep cerebral white matter in PVL. No data are yet available as to whether there is any genetic predisposition to PVL or to its neurological sequelae. Multiple sclerosis (MS) is an inflammatory white matter disease that often begins in young adulthood, causes multifocal destruction of mature oligodendroglia and of axons, and eventually leads to substantial cumulative neurological disability. Certain genetic polymorphisms contribute to susceptibility to MS, and adaptive immune responses to myelin-associated self antigens, or to exogenous antigens that mimic these self antigens, play a central role in the pathophysiology of this disease.