Liposome-Mediated Anti-Viral Drug Delivery Across Blood-Brain Barrier: Can Lipid Droplet Target Be Game Changers?

Lipid droplets (LDs) are subcellular organelles secreted from the endoplasmic reticulum (ER) that play a major role in lipid homeostasis. Recent research elucidates additional roles of LDs in cellular bioenergetics and innate immunity. LDs activate signaling cascades for interferon response and secretion of pro-inflammatory cytokines. Since balanced lipid homeostasis is critical for neuronal health, LDs play a crucial role in neurodegenerative diseases. RNA viruses enhance the secretion of LDs to support various phases of their life cycle in neurons which further leads to neurodegeneration. Targeting the excess LD formation in the brain could give us a new arsenal of antiviral therapeutics against neuroviruses. Liposomes are a suitable drug delivery system that could be used for drug delivery in the brain by crossing the Blood-Brain Barrier. Utilizing this, various pharmacological inhibitors and non-coding RNAs can be delivered that could inhibit the biogenesis of LDs or reduce their sizes, reversing the excess lipid-related imbalance in neurons. Liposome-Mediated Antiviral Drug Delivery Across Blood-Brain Barrier. Developing effective antiviral drug is challenging and it doubles against neuroviruses that needs delivery across the Blood-Brain Barrier (BBB). Lipid Droplets (LDs) are interesting targets for developing antivirals, hence targeting LD formation by drugs delivered using Liposomes can be game changers.

Beta-coronaviruses exploit cellular stress responses by modulating TFEB and TFE3 activity.

Beta-coronaviruses have emerged as a severe threat to global health. Undercovering the interplay between host and beta-coronaviruses is essential for understanding disease pathogenesis and developing efficient treatments. Here we report that the transcription factors TFEB and TFE3 translocate from the cytosol to the nucleus in response to beta-coronavirus infection by a mechanism that requires activation of calcineurin phosphatase. In the nucleus, TFEB and TFE3 bind to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators is significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 is sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 results in increased cellular survival of infected cells but also in reduced lysosomal exocytosis and decreased viral infectivity. These results suggest a central role of TFEB and TFE3 in cellular response to beta-coronavirus infection.

Vesicle-Cloaked Rotavirus Clusters are Environmentally Persistent and Resistant to Free Chlorine Disinfection.

Recent discovery of vesicle-cloaked virus clusters (i.e., viral vesicles) has greatly challenged the central paradigm of viral transmission and infection as a single virion. To understand the environmental transmission of viral vesicles, we used an in vivo model to investigate their environmental persistence and engineering control by disinfection. Murine rotavirus vesicles maintained both their integrity and infectivity after incubation in filtered freshwater and wastewater for at least 7 days, with 24.5-27.5% of the vesicles still intact at 16 weeks after exposure to both waters. Free chlorine disinfection at a dosage of 13.3 mg min L-1 did not decompose murine rotavirus vesicles, and it was much less effective in inactivating rotaviruses inside vesicles than free rotaviruses based on the quantification of rotavirus shedding in mouse stool and rotavirus replication in small intestines. Rotavirus vesicles may be more environmentally transmissible than free rotaviruses regardless of disinfection. Vesicle-mediated en bloc transmission could be responsible for vesicles' resistance to disinfection due to an increased multiplicity of infection and/or genetic recombination or reassortment to promote infection. Our work highlights the environmental, biological, and public health significance of viral vesicles, and the findings call for urgent action in advancing disinfection for pathogen control.

Emerging Pathogenic Unit of Vesicle-Cloaked Murine Norovirus Clusters is Resistant to Environmental Stresses and UV254 Disinfection.

An individual virion was long believed to act as an independent infectious unit in virology, until the recent discovery of vesicle-cloaked virus clusters which has greatly challenged this central paradigm. Vesicle-cloaked virus clusters (also known as viral vesicles) are phospholipid-bilayer encapsulated fluid sacs that contain multiple virions or multiple copies of viral genomes. Norovirus is a global leading causative agent of gastroenteritis, and the reported prevalence of vesicle-cloaked norovirus clusters in stool has raised concerns whether the current disinfection, sanitation, and hygiene practices can effectively control environmental pollution by these pathogenic units. In this study, we have demonstrated that vesicle-cloaked murine norovirus (MNV-1) clusters were highly persistent under temperature variation (i.e., freeze-thaw) and they were partially resistant to detergent decomposition. MNV-1 vesicles were 1.89-3.17-fold more infectious in vitro than their free virus counterparts. Most importantly, MNV-1 vesicles were up to 2.16-times more resistant to UV254 disinfection than free MNV-1 at a low viral load in vitro. Interestingly, with the increase of the viral load, free MNV-1 and MNV-1 vesicles showed equivalent resistance to UV254 disinfection. We show that the increased multiplicity of infection provided by vesicles is in part responsible for these attributes. Our study, for the first time, sheds light on the environmental behavior of vesicle-cloaked virus clusters as unique emerging pathogenic units. Our study highlights the need to revisit current paradigms of disinfection, sanitation, and hygiene practices for protecting public health.

ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Norovirus infection causes acute self-resolving diarrhea in wild-type neonatal mice.

Human noroviruses are the leading cause of severe childhood diarrhea worldwide, yet we know little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a norovirus small animal disease model in wild-type mice. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response.

Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission.

In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.

Chandipura Virus Induced Neuronal Apoptosis via Calcium Signaling Mediated Oxidative Stress.

Chandipura Virus (CHPV) a negative-stranded RNA virus belonging to the Rhabdoviridae family, has been previously reported to bring about neuronal apoptosis by stimulating oxidative stress. Our in silico data suggested the involvement of Angiotensin II in intracellular Ca2+ secretion within CHPV infected cells that further lead to enhancement of ROS level and mitochondrial dysfunction. ROS is also known to phosphorylate p38 that leads to neuronal apoptosis through FasL-FADD pathway during CHPV infection. Minocycline a broad-spectrum antibiotic well-known for its anti-oxidative and anti-inflammatory role was used in the present study to investigate its efficacy against CHPV. The results obtained from the present study showed minocycline to be effective in mitigating the levels of cytoplasmic Ca2+, ROS, phosphorylation of p38 molecules and hence cellular apoptosis. Thus minocycline apart from being an anti-inflammatory and anti-oxidative agent, our study showed that minocycline has an additional Ca2+ chelation activity.

Neuropathogenesis by Chandipura virus: An acute encephalitis syndrome in India.

Chandipura virus (CHPV) has been contributing to the rising number of premature deaths due to acute encephalitis syndrome for over a decade in India. CHPV belongs to the family Rhabdoviridae. Neuropathogenesis of CHPV has been well established but the exact route of entry into the central nervous system (CNS) and the triggering factor for neuronal death are still unknown. Rabies virus and vesicular stomatitis virus, which are related closely to CHPV, enter the CNS retrogradely from peripheral or olfactory neurons. Disruption of the blood-brain barrier has also been connoted in the entry of CHPV into the CNS. CHPV upon entering the neurons triggers cellular stress factors and release of reactive oxygen species (ROS). The stress granules produced in response to cellular stress have been implicated in viral replication and ROS generation, which stimulates neuronal death. Both these phenomena cohesively explain the neuropathogenesis and neurodegeneration following CHPV infection.

Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses.

Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1's role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain.

Microglial activation induces neuronal death in Chandipura virus infection.

Neurotropic viruses induce neurodegeneration either directly by activating host death domains or indirectly through host immune response pathways. Chandipura Virus (CHPV) belonging to family Rhabdoviridae is ranked among the emerging pathogens of the Indian subcontinent. Previously we have reported that CHPV induces neurodegeneration albeit the root cause of this degeneration is still an open question. In this study we explored the role of microglia following CHPV infection. Phenotypic analysis of microglia through lectin and Iba-1 staining indicated cells were in an activated state post CHPV infection in cortical region of the infected mouse brain. Cytokine Bead Array (CBA) analysis revealed comparatively higher cytokine and chemokine levels in the same region. Increased level of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), Nitric Oxide (NO) and Reactive Oxygen species (ROS) in CHPV infected mouse brain indicated a strong inflammatory response to CHPV infection. Hence it was hypothesized through our analyses that this inflammatory response may stimulate the neuronal death following CHPV infection. In order to validate our hypothesis supernatant from CHPV infected microglial culture was used to infect neuronal cell line and primary neurons. This study confirmed the bystander killing of neurons due to activation of microglia post CHPV infection.

HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NFκB determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks.

Graph theoretic network analysis reveals protein pathways underlying cell death following neurotropic viral infection.

Complex protein networks underlie any cellular function. Certain proteins play a pivotal role in many network configurations, disruption of whose expression proves fatal to the cell. An efficient method to tease out such key proteins in a network is still unavailable. Here, we used graph-theoretic measures on protein-protein interaction data (interactome) to extract biophysically relevant information about individual protein regulation and network properties such as formation of function specific modules (sub-networks) of proteins. We took 5 major proteins that are involved in neuronal apoptosis post Chandipura Virus (CHPV) infection as seed proteins in a database to create a meta-network of immediately interacting proteins (1(st) order network). Graph theoretic measures were employed to rank the proteins in terms of their connectivity and the degree upto which they can be organized into smaller modules (hubs). We repeated the analysis on 2(nd) order interactome that includes proteins connected directly with proteins of 1(st) order. FADD and Casp-3 were connected maximally to other proteins in both analyses, thus indicating their importance in neuronal apoptosis. Thus, our analysis provides a blueprint for the detection and validation of protein networks disrupted by viral infections.

Chandipura virus perturbs cholesterol homeostasis leading to neuronal apoptosis.

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) induces neuronal death through the Fas-mediated extrinsic apoptosis pathway. What propels this apoptosis remains unclear, although oxysterols have been reported to be key players in neurodegeneration. In our study of CHPV-infected brain samples, we observed over-expression of genes such as apolipoprotein E, Cyp46a1, Srebf-1 and Nsdhl. This backs up the hypothesis that CHPV replication demands cholesterol that is supplied by apolipoprotein E through low density lipid receptors, lipid metabolism being pivotal for viral replication. We were able to illustrate this with over-expression of low density lipid receptors in CHPV-infected neurons. An upsurge of cholesterol concentration has been observed in neurons, triggering the expression of Cyp46a1 enzyme and culminating into the conversion of cholesterol to 24(S)-hydroxycholesterol. Increased 24(S)-hydroxycholesterol concentration is toxic to neurons, propelling neuronal apoptosis through the Fas-mediated extrinsic apoptosis pathway. For the first time, perturbation of cholesterol homeostasis in brain is shown to be utilized by the viruses for both maturation and the release of its matured virions outside the cells for continuous neuropathogenesis.

RIG-I knockdown impedes neurogenesis in a murine model of Japanese encephalitis.

Retinoic acid inducible gene I (RIG-I) is a well established pattern recognition receptor (PRR) in neurons infected with Japanese encephalitis virus (JEV) as reported previously from our laboratory. Japanese encephalitis (JE) virus infection in brain has been shown to decrease the proliferation of neural stem/progenitor cells (NSPCs) which has its implications in neurological sequelae in JE survivors. We have found that ablation of RIG-I both in vivo and in vitro models results in significant decrease in NSPC proliferation post JEV infection. We hypothesize that knockdown of RIG-I diminishes the expression of antiviral molecules resulting in an increase in viral replication, which in turn results in enhancement of the expression of cell cycle inhibitors, hence affecting the proliferation of NSPCs.

Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection.

In this study we have reported the in vivo proteomic changes during Japanese Encephalitis Virus (JEV) infection in combination with in vitro studies which will help in the comprehensive characterization of the modifications in the host metabolism in response to JEV infection. We performed a 2-DE based quantitative proteomic study of JEV-infected mouse brain as well as mouse neuroblastoma (Neuro2a) cells to analyze the host response to this lethal virus. 56 host proteins were found to be differentially expressed post JEV infection (defined as exhibiting ≥ 1.5-fold change in protein abundance upon JEV infection). Bioinformatics analyses were used to generate JEV-regulated host response networks which reported that the identified proteins were found to be associated with various cellular processes ranging from intracellular protein transport, cellular metabolism and ER stress associated unfolded protein response. JEV was found to invade the host protein folding machinery to sustain its survival and replication inside the host thereby generating a vigorous unfolded protein response, subsequently triggering a number of pathways responsible for the JEV associated pathologies. The results were also validated using a human cell line to correlate them to the human response to JEV. The present investigation is the first report on JEV-host interactome in in vivo model and will be of potential interest for future antiviral research in this field.

Chandipura virus induces neuronal death through Fas-mediated extrinsic apoptotic pathway.

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.

Network medicine in drug design: implications for neuroinflammation.

Neuroinflammation is a general innate defensive response to neurotropic pathogens, neurodegenerative diseases or brain injuries, brought about by active proinflammatory signaling by the glial cells (microglia and astrocytes). Because these inflammatory signaling pathways cross-talk with each other, drug targeting at any particular intermediate molecule is not effective. Network medicine is a network theory inspired approach in drug design, whereby various mathematical models are applied to identify plausible nodes within a signaling pathway simulated network important for drug targeting. There are many techniques involved in network medicine study; in this article we concentrate on the 'prioritization of protein clusters' responsible for a certain disorder. This approach aims to bring down the expenditure of resources of initial drug targeting against a complex pathological reaction, such as neuroinflammation, and also questions the cause at the molecular level.