Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.

Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.

SARS-CoV-2 infection induces thymic atrophy mediated by IFN-γ in hACE2 transgenic mice.

Pathogenic infections cause thymic atrophy, perturb thymic T-cell development, and alter immunological response. Previous studies reported dysregulated T-cell function and lymphopenia in coronavirus disease-19 (COVID-19). However, immunopathological changes in the thymus associated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report that SARS-CoV-2 infects thymocytes, and induces CD4+CD8+ (double positive; DP) T-cell apoptosis leading to thymic atrophy and loss of peripheral TCR repertoire in K18-hACE2 transgenic mice. Infected thymus led to increased CD44+CD25- T-cells, indicating an early arrest in the T-cell maturation pathway. Thymic atrophy was notably higher in male hACE2-Tg mice than in females and involved an upregulated de-novo synthesis pathway of thymic glucocorticoid. Further, IFN-γ was crucial for thymic atrophy, as anti-IFN-γ -antibody neutralization blunted thymic involution. Therapeutic use of Remdesivir also rescued thymic atrophy. While the Omicron variant and its sub-lineage BA.5 variant caused marginal thymic atrophy, the delta variant of SARS-CoV-2 exhibited severe thymic atrophy characterized by severely depleted DP T-cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore the thymic maturation pathway of T-cells. Together, we report SARS-CoV-2-associated thymic atrophy resulting from impaired T-cell maturation pathway which may contribute to dyregulated T cell response during COVID-19.

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy.

Japanese encephalitis virus (JEV) pathogenesis is driven by a combination of neuronal death and neuroinflammation. We tested 42 FDA-approved drugs that were shown to induce autophagy for antiviral effects. Four drugs were tested in the JE mouse model based on in vitro protective effects on neuronal cell death, inhibition of viral replication, and anti-inflammatory effects. The antipsychotic phenothiazines Methotrimeprazine (MTP) & Trifluoperazine showed a significant survival benefit with reduced virus titers in the brain, prevention of BBB breach, and inhibition of neuroinflammation. Both drugs were potent mTOR-independent autophagy flux inducers. MTP inhibited SERCA channel functioning, and induced an adaptive ER stress response in diverse cell types. Pharmacological rescue of ER stress blocked autophagy and antiviral effect. MTP did not alter translation of viral RNA, but exerted autophagy-dependent antiviral effect by inhibiting JEV replication complexes. Drug-induced autophagy resulted in reduced NLRP3 protein levels, and attenuation of inflammatory cytokine/chemokine release from infected microglial cells. Our study suggests that MTP exerts a combined antiviral and anti-inflammatory effect in JEV infection, and has therapeutic potential for JE treatment.

Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections.

BACKGROUND: Activated platelets secrete platelet factor 4 (PF4), which contributes to viral pathogenesis. Recently, we reported the proviral role of PF4 in replication of closely related flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV). OBJECTIVES: This study aimed to investigate the detailed mechanism of PF4-mediated virus replication. METHODS: PF4-/- or wild-type (WT) mice were infected with JEV, and host defense mechanisms, including autophagic/interferon (IFN) responses, were assessed. WT mice were pretreated with the CXCR3 antagonist AMG487 that inhibits PF4:CXCR3 pathway. This pathway was tested in PF4-/- monocytes infected with DENV or in monocytes isolated from patients with DENV infection. RESULTS: PF4-/- mice infected with JEV showed reduced viral load and improved brain inflammation and survival. PF4-/- mice synthesized more IFN-α/ß with higher expression of phosphorylated IRF3 in the brain. PF4 treatment decreased IRF-3/7/9 and IFN-α/ß expression and suppressed autophagic LC3-II flux and lysosomal degradation of viral proteins in JEV-infected cells. PF4 increased the expression of P-mTOR, P-p38, and P-ULK1Ser757 and decreased expression of LC3-II. Decreased autophagosome-lysosome fusion in turn promoted DENV2 replication. The above processes were reversed by AMG487. Uninfected PF4-/- monocytes showed elevated LC3-II and autophagosome-lysosome fusion. Microglia of JEV-infected PF4-/- mice exhibited elevated LC3-II inversely related to viral load. Similarly, monocytes from PF4-/- mice showed reduced infection by DENV2. In patients with DENV infection, higher plasma PF4 and viral load were inversely correlated with LC3-II, LAMP-1, and lysosomal degradation of DENV-NS1 in monocytes during the febrile phase. CONCLUSION: These studies suggest that PF4 deficiency or inhibition of the PF4:CXCR3 pathway prevents JEV and DENV infection. The studies also highlight the PF4:CXCR3 axis as a potential target to develop treatment regimens against flaviviruses.

Japanese Encephalitis Virus-Infected Cells.

RNA virus infections have been a leading cause of pandemics. Aided by global warming and increased connectivity, their threat is likely to increase over time. The flaviviruses are one such RNA virus family, and its prototypes such as the Japanese encephalitis virus (JEV), Dengue virus, Zika virus, West Nile virus, etc., pose a significant health burden on several endemic countries. All viruses start off their life cycle with an infected cell, wherein a series of events are set in motion as the virus and host battle for autonomy. With their remarkable capacity to hijack cellular systems and, subvert/escape defence pathways, viruses are able to establish infection and disseminate in the body, causing disease. Using this strategy, JEV replicates and spreads through several cell types such as epithelial cells, fibroblasts, monocytes and macrophages, and ultimately breaches the blood-brain barrier to infect neurons and microglia. The neurotropic nature of JEV, its high burden on the paediatric population, and its lack of any specific antivirals/treatment strategies emphasise the need for biomedical research-driven solutions. Here, we highlight the latest research developments on Japanese encephalitis virus-infected cells and discuss how these can aid in the development of future therapies.

Guanylate-binding proteins in virus infection.

Guanylate-binding proteins (GBPs) are immune GTPases that are induced in response to interferon stimulation/pathogen infection. These proteins arose early in evolution and have multiple physiological roles ranging from tumor suppression to anti-microbial functions. While several studies describe their mechanistic role in the lysis of bacteria/pathogen vacuole, and activation of the inflammasome, their functions in viral infections are only just emerging. The role of the GBPs in virus infections is multifaceted, being both dependent on and independent of GTP binding/hydrolysis and isoprenylation. Diverse antiviral roles are documented such as inhibition of viral RNA/protein synthesis, block of viral envelope glycoprotein processing, and targeting viral protein for degradation. Not surprisingly, several viral proteins bind to specific GBPs and antagonize their antiviral effects. While recruitment of GBP1, Gbp1, Gbp2 on the virus replication complex has been reported, the functional implications of this are not entirely clear. Furthermore, their role in interferon and inflammation activation during virus infection are contradictory, with reports of both positive and negative regulation. Here, we discuss the emerging functional roles of GBPs in virus infections.

Human Guanylate-Binding Protein 1 Positively Regulates Japanese Encephalitis Virus Replication in an Interferon Gamma Primed Environment.

RNA virus infection triggers interferon (IFN) receptor signaling, leading to the activation of hundreds of interferon-stimulated genes (ISGs). Guanylate-binding proteins (GBPs) belong to one such IFN inducible subfamily of guanosine triphosphatases (GTPases) that have been reported to exert broad anti-microbial activity and regulate host defenses against several intracellular pathogens. Here, we investigated the role of human GBP1 (hGBP1) in Japanese encephalitis virus (JEV) infection of HeLa cells in both an IFNγ unprimed and primed environment. We observed enhanced expression of GBP1 both at transcript and protein levels upon JEV infection, and GBP1 association with the virus replication membranes. Depletion of hGBP1 through siRNA had no effect on JEV replication or virus induced cell death in the IFNγ unprimed environment. IFNγ stimulation provided robust protection against JEV infection. Knockdown of GBP1 in the primed environment upregulated expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and significantly reduced JEV replication. Depletion of GBP1 in an IFNγ primed environment also inhibited virus replication in human neuroblastoma SH-SH5Y cells. Our data suggests that in the presence of IFNγ, GBP1 displays a proviral role by inhibiting innate immune responses to JEV infection.

Japanese Encephalitis Virus NS4A Protein Interacts with PTEN-Induced Kinase 1 (PINK1) and Promotes Mitophagy in Infected Cells.

The nonstructural protein 4A (NS4A) of flaviviruses has been implicated as a "central organizer" of the membrane-bound replication complex during virus replication. However, its role in the host responses to virus infection is not understood. Using the yeast-two-hybrid library screen, we identified a multitude of host proteins interacting with the Japanese encephalitis virus (JEV) NS4A protein. Several of these interacting proteins are known to localize to the mitochondria. One of these proteins was PTEN-induced kinase 1 (PINK1), a serine/threonine-protein kinase known for its role in mitophagy. Here, we demonstrate the JEV-NS4A localization to the mitochondria and its interaction with PINK1 in Huh7 cells during JEV infection. The JEV-infected cells showed an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We present data showing that JEV-NS4A alone was sufficient to induce mitophagy. Interference with mitochondrial fragmentation and mitophagy resulted in reduced virus propagation. Overall, our study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein. IMPORTANCE The JEV-infected mammalian cells show an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We show that the NS4A protein of JEV localized to the mitochondria and interacted with PINK1 in Huh7 cells during infection with the virus and demonstrate that JEV-NS4A alone is sufficient to induce mitophagy. The study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein.

Studying Autophagy Using a TMT-Based Quantitative Proteomics Approach.

Maintenance of cellular homeostasis through regulated degradation of proteins and organelles is a defining feature of autophagy. This process itself is tightly regulated in a series of well-defined biochemical reactions governed largely by the highly conserved ATG protein family. Given its crucial role in regulating protein levels under both basal and stress conditions such as starvation and infection, genetic or pharmacological perturbation of autophagy results in massive changes in the cellular proteome and impacts nearly every biological process. Therefore, studying autophagy perturbations at a global scale assumes prime importance. In recent years, quantitative mass spectrometry (MS)-based proteomics has emerged as a powerful approach to explore biological processes through global proteome quantification analysis. Tandem mass tag (TMT)-based MS proteomics is one such robust quantitative technique that can examine relative protein abundances in multiple samples (parallel multiplexing). Investigating autophagy through TMT-based MS approach can give great insights into autophagy-regulated biological processes, protein-protein interaction networks, spatiotemporal protein dynamics, and identification of new autophagy substrates. This chapter provides a detailed protocol for studying the impact of a dysfunctional autophagy pathway on the cellular proteome and pathways in a healthy vs. disease (virus infection) condition using a 16-plex TMT-based quantitative proteomics approach. We also provide a pipeline on data processing and analysis using available web-based tools.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts.

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses.

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC-I antigen presentation and stress granule signaling are enhanced in IRGM-deficient cells, indicating a robust cell-intrinsic antiviral immune state. Consistently, IRGM-depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS-CoV-2, CHIKV, and Zika virus.

Retinoic Acid-Inducible Gene I-Like Receptors Activate Snail To Limit RNA Viral Infections.

Retinoic acid-inducible gene I-like receptors (RLRs) are important cytosolic pattern recognition receptors (PRRs) that sense viral RNA before mounting a response leading to the activation of type I IFNs. Several viral infections induce epithelial-mesenchymal transition (EMT), even as its significance remains unclear. Here, we show that EMT or an EMT-like process is a general response to viral infections. Our studies identify a previously unknown mechanism of regulation of an important EMT-transcription factor (EMT-TF) Snail during RNA viral infections and describe its possible implication. RNA viral infections, poly(I·C) transfection, and ectopic expression of RLR components induced Snail levels, indicating that RLR pathway could regulate its expression. Detailed examination using mitochondrial antiviral signaling protein knockout (MAVS-KO) cells established that MAVS is essential in this regulation. We identified two interferon-stimulated response elements (ISREs) in the SNAI1 promoter region and demonstrated that they are important in its transcriptional activation by phosphorylated IRF3. Increasing the levels of Snail activated RLR pathway and dramatically limited replication of the RNA viruses dengue virus, Japanese encephalitis virus (JEV), and vesicular stomatitis virus, pointing to their antiviral functions. Knockdown of Snail resulted in a considerable increase in the JEV titer, validating its antiviral functions. Finally, transforming growth factor ß-mediated IFNB activation was dependent on Snail levels, confirming its important role in type I IFN activation. Thus, EMT-TF Snail is transcriptionally coregulated with type I IFN by RLRs and, in turn, promotes the RLR pathway, further strengthening the antiviral state in the cell. Our work identified an interesting mechanism of regulation of Snail that demonstrates potential coregulation of multiple innate antiviral pathways triggered by RLRs. Identification of antiviral functions of Snail also provides an opportunity to expand the sphere of RLR signaling. IMPORTANCE RLRs sense viral genomic RNA or the double-stranded RNA intermediates and trigger the activation of type I IFNs. Snail transcription factor, commonly associated with epithelial-mesenchymal transition (EMT), has been reported to facilitate EMT in several viral infections. Many of these reports are based on oncoviruses, leading to the speculation that EMT induced during infection is an important factor in the oncogenesis triggered by these infections. However, our studies reveal that EMT or EMT-like processes during viral infections have important functions in antiviral response. We have characterized a new mechanism of transcriptional regulation of Snail by IRF3 through interferon-stimulated response elements in their promoters, and this finding could have importance in nonviral contexts as well. We also identify that EMT-TF Snail promotes antiviral status of the infected cells through the RLR pathway. This study characterizes a new regulatory mechanism of activation of Snail and establishes its unidentified function in antiviral response.

Japanese Encephalitis Virus Infected Human Monocyte-Derived Dendritic Cells Activate a Transcriptional Network Leading to an Antiviral Inflammatory Response.

A comprehensive understanding of the human immune response to virus infection is imperative for developing effective therapies, antivirals, and vaccines. Dendritic cells (DCs) are among the first cells to encounter the virus and are also key antigen-presenting cells that link the innate and adaptive immune system. In this study, we focus on the human immune response to the mosquito-borne Japanese encephalitis virus (JEV), which is the leading cause of virus-induced encephalitis in south-east Asia and has the potential to become a global pathogen. We describe the gene regulatory circuit of JEV infection in human monocyte-derived DCs (moDCs) along with its functional validation. We observe that JEV can productively infect human moDCs leading to robust transcriptional activation of the interferon and NF-κB-mediated antiviral and inflammatory pathways. This is accompanied with DC maturation and release of pro-inflammatory cytokines and chemokines TNFα, IL-6, IL-8, IL-12, MCP-1. and RANTES. JEV-infected moDCs activated T-regulatory cells (Tregs) in allogenic mixed lymphocyte reactions (MLR) as seen by upregulated FOXP3 mRNA expression, suggestive of a host response to reduce virus-induced immunopathology. The virus also downregulated transcripts involved in Peroxisome Proliferator Activated Receptor (PPAR) signalling and fatty acid metabolism pathways suggesting that changes in cellular metabolism play a crucial role in driving the DC maturation and antiviral responses. Collectively, our data describe and corroborate the human DC transcriptional network that is engaged upon JEV sensing.

Pathobiology of Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) is a flavivirus, spread by the bite of carrier Culex mosquitoes. The subsequent disease caused is Japanese encephalitis (JE), which is the leading global cause of virus-induced encephalitis. The disease is predominant in the entire Asia-Pacific region with the potential of global spread. JEV is highly neuroinvasive with symptoms ranging from mild fever to severe encephalitis and death. One-third of JE infections are fatal, and half of the survivors develop permanent neurological sequelae. Disease prognosis is determined by a series of complex and intertwined signaling events dictated both by the virus and the host. All flaviviruses, including JEV replicate in close association with ER derived membranes by channelizing the protein and lipid components of the ER. This leads to activation of acute stress responses in the infected cell-oxidative stress, ER stress, and autophagy. The host innate immune and inflammatory responses also enter the fray, the components of which are inextricably linked to the cellular stress responses. These are especially crucial in the periphery for dendritic cell maturation and establishment of adaptive immunity. The pathogenesis of JEV is a combination of direct virus induced neuronal cell death and an uncontrolled neuroinflammatory response. Here we provide a comprehensive review of the JEV life cycle and how the cellular stress responses dictate the pathobiology and resulting immune response. We also deliberate on how modulation of these stress pathways could be a potential strategy to develop therapeutic interventions, and define the persisting challenges.

Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets.

Microtubule-associated protein 1 light chain 3 (MAP1LC3) is a protein with a well-defined function in autophagy, but still incompletely understood roles in several other autophagy-independent processess. Studies have shown MAP1LC3 is a host-dependency factor for the replication of several viruses. Japanese encephalitis virus (JEV), a neurotropic flavivirus, replicates on ER-derived membranes that are marked by autophagosome-negative non-lipidated MAP1LC3 (LC3-I). Depletion of LC3 exerts a profound inhibition on virus replication and egress. Here, we further characterize the role of LC3 in JEV replication, and through immunofluorescence and immunoprecipitation show that LC3-I interacts with the virus capsid protein in infected cells. This association was observed on capsid localized to both the replication complex and lipid droplets (LDs). JEV infection decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication. This capsid-LC3 interaction was independent of the autophagy adaptor protein p62/Sequestosome 1 (SQSTM1). Further, no association of capsid was seen with the Gamma-aminobutyric acid receptor-associated protein family, suggesting that this interaction was specific for LC3. High-resolution protein-protein docking studies identified a putative LC3-interacting region in capsid, 56FTAL59, and other key residues that could mediate a direct interaction between the two proteins.

Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells.

Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

Synergistic antibacterial and anti-biofilm activity of nisin like bacteriocin with curcumin and cinnamaldehyde against ESBL and MBL producing clinical strains.

Bacteriocins are small peptides that can inhibit the growth of a diverse range of microbes. There is a need to identify bacteriocins that are effective against biofilms of resistant clinical strains. The present study focussed on the efficacy of purified nisin like bacteriocin-GAM217 against extended spectrum ß-lactamase (ESBL) and metallo-beta-lactamase (MBL) producing clinical strains. Bacteriocin-GAM217 when combined with curcumin and cinnamaldehyde, synergistically enhanced antibacterial activity against planktonic and biofilm cultures of Staphylococcus epidermidis and Escherichia coli. Bacteriocin-GAM217 and phytochemical combinations inhibited biofilm formation by >80%, and disrupted the biofilm for selected ESBL and MBL producing clinical strains. The anti-adhesion assay showed that these combinatorial compounds significantly lowered the attachment of bacteria to Vero cells and that they elicited membrane permeability and rapid killing as viewed by confocal microscopy. This study demonstrates that bacteriocin-GAM217 in combination with phytochemicals can be a potential anti-biofilm agent and thus has potential for biomedical applications.

Quantitative Proteome Analysis of Atg5-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome.

Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5-/- MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor ß (TGF-ß) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5-/- MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome.IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway (Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells.