Sudhanshu Vrati - Academia.edu (original) (raw)

Papers by Sudhanshu Vrati

Autophagy is a conserved intracellular degradation pathway that is essential for maintaining cell... more Autophagy is a conserved intracellular degradation pathway that is essential for maintaining cellular homeostasis. Given its critical role in several disease conditions, recent studies are focussed on identifying drugs/small molecules with autophagy modulating capacity for potential clinical applications. Here, we describe the development and characterisation of a quantitative image-based high content screening platform for autophagy flux measurements using the human melanoma A375 cell line that stably expresses the GFP-LC3-RFP probe. The GFP-LC3 is incorporated into autophagosomes, while RFP serves an internal control. The GFP/RFP fluorescence intensity ratio gives an accurate indication of autophagy induction (low ratio) vs blockage of autophagy flux (high ratio), and was validated with the autophagy inducer Torin1 and inhibitor Bafilomycin A1. This assay was used to screen the Spectrum collection library comprising of 2560 compounds, to identify autophagy modulators. In addition to known autophagy effectors, several novel autophagy inducers and inhibitors were identified in our study. Further three FDA approved drugs that are widely used in skin-care products: Avobenzone, Guaiazulene and Tioxolone, were validated as potent autophagy inducers that function in an mTOR independent manner.

Scientific Reports, Dec 4, 2015

Flavivirus infection causes host cell death by initiation of an unfolded protein response (UPR). ... more Flavivirus infection causes host cell death by initiation of an unfolded protein response (UPR). UPR is initiated following activation of three ER-membrane resident sensors, PERK, IRE1α and ATF6, which are otherwise kept inactive through association with the ER-chaperone GRP78. Activation precedes cellular and molecular changes that act to restore homeostasis but might eventually initiate apoptosis. These changes involve influencing function of multiple genes by either transcriptional or post-transcriptional or post-translational mechanisms. Transcriptional control includes expression of transcription factor cascades, which influence cognate gene expression. Malat1 is a long non-coding RNA which is over-expressed in many human oncogenic tissues and regulates cell cycle and survival. In this report, for the first time we show activation of Malat1 following infection by two flaviviruses, both of which activate the UPR in host cells. The temporal kinetics of expression was restricted to later time points. Further, Malat1 was also activated by pharmacological inducer of UPR, to a similar degree. Using drugs that specifically inhibit or activate the PERK or IRE1α sensors, we demonstrate that signalling through the PERK axis activates this expression, through a transcriptional mechanism. To our knowledge, this is the first report of an UPR pathway regulating the expression of an lncRNA. Flaviviridae constitutes an important group of human pathogenic virus responsible for extensive death and debilitation in different parts of the world 1. In addition to innate anti-viral immune pathways the infection following multiple flaviviruses activates an unfolded protein response (UPR) through saturation of the protein folding capacity in host cell endoplasmic reticulum (ER) 2-4. The induction of UPR has been suggested as the principal cause behind the apoptotic cell death observed in infected cells. UPR is induced following infection by a wide variety of viruses, many of which have evolved to regulate the downstream signalling 5. The primary objective of the cellular changes observed during an UPR is to restore homeostasis, failing which the cell is committed to an apoptotic death. Unfolded/malfolded proteins that accumulate in the ER lumen form stable complexes with the ER chaperone HSPA5/Bip/GRP78. Under homeostatic conditions, GRP78 remains associated with the ER-lumen resident domain of three ER-membrane resident trans-membrane protein sensors, namely PKR-like ER Kinase (PERK), Inositol-responsive enzyme 1 (IRE1) and Activating transcription factor 6 (ATF6). A continued association with GRP78 molecules maintains these sensors in a dormant state. The accumulated unfolded proteins compete with these

Virology, Jun 1, 1996

The genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses... more The genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses. To facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. The cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5 termini. Transfection of the linear DNA into cells which supported replication produced infectious virus. Mutation of a unique SalI site at the right-hand end of the genome disrupted reading frames of unknown function without affecting virus rescue, identifying this region as nonessential for replication in vitro. A 20-bp oligonucleotide was also inserted into the short intergenic region between the pVIII and the fiber sequences, identifying a second site for gene insertion. These studies will facilitate the development of OAV as a gene transfer vector.

Methods in molecular biology, 2022

Maintenance of cellular homeostasis through regulated degradation of proteins and organelles is a... more 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.

Biochemical and Biophysical Research Communications, Jul 1, 2018

Diphenyleneiodonium (DPI) and N-acetyl-L-cysteine (NAC), two widely used anti-oxidants, were empl... more Diphenyleneiodonium (DPI) and N-acetyl-L-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes.

Archives of Virology, Mar 1, 1992

PLOS Neglected Tropical Diseases, Feb 2, 2017

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provid... more Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β-T cells (TCRβ-null) are highly susceptible and die over 10-18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the bloodbrain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

Scientific Reports, Aug 18, 2017

Stringent regulation of antiviral signaling and cellular autophagy is critical for the host respo... more Stringent regulation of antiviral signaling and cellular autophagy is critical for the host response to virus infection. However, little is known how these cellular processes are regulated in the absence of type I interferon signaling. Here, we show that ATF3 is induced following Japanese encephalitis virus (JEV) infection, and regulates cellular antiviral and autophagy pathways in the absence of type I interferons in mouse neuronal cells. We have identified new targets of ATF3 and show that it binds to the promoter regions of Stat1, Irf9, Isg15 and Atg5 thereby inhibiting cellular antiviral signaling and autophagy. Consistent with these observations, ATF3-depleted cells showed enhanced antiviral responses and induction of robust autophagy. Furthermore, we show that JEV replication was significantly reduced in ATF3-depleted cells. Our findings identify ATF3 as a negative regulator of antiviral signaling and cellular autophagy in mammalian cells, and demonstrate its important role in JEV life cycle. Viruses are arduous pathogens that pose a unique challenge to our immune system as they are composed of the host-derived molecules. However, viral nucleic acids possess unique features distinguishing them from the host which have possibly led to the evolution of Pattern Recognition Receptors (PRRs) for their detection. Among the PRRs, RIG-I-like receptors (RLRs) are ubiquitous cytosolic detectors which play an integral role in antiviral responses 1. Following the detection of viral infections, the PRR-initiated antiviral signaling rapidly induces the production of type 1 interferons (IFNa and IFNb) and other pro-inflammatory cytokines. The IFNs once released into the extracellular milieu bind to their respective membrane-bound receptors and initiate downstream signaling leading to the modulation of expression of a cohort of antiviral genes termed as Interferon Sensitive Genes (ISGs) 2. The IFNs can potentially act in an autocrine or paracrine manner to subvert an existing viral infection or induce a pre-emptive antiviral state, respectively. Both the primary response (PRR activation followed by IFN synthesis) and the secondary response (IFN-receptor interaction to modulate the ISG expression) are driven by a dedicated family of transcription factors (TFs). The primary response is mainly driven by Interferon Regulatory Factor (IRF) family of TFs while the secondary response depends on the activity of the STAT proteins as part of the JAK-STAT pathway 3, 4. Binding of IFNs to their receptors leads to receptor dimerization followed by the activation of IRF and STAT family of transcription factors. STAT1 and STAT2 dimerize and interact with IRF9 to form the Interferon-Stimulated Gene Factor 3 (ISGF3) complex 5. This complex then translocates to the nucleus and binds to the conserved Interferon-Stimulated Response Elements (ISREs) resulting in the induction of various ISGs. Apart from the induction of ISGs, type 1 IFN signaling plays a pivotal role in regulating other cellular processes like autoimmunity 6 , cancer 7 and autophagy 8, 9. Autophagy is a highly conserved phenomenon in which cells digest their own cytoplasmic content in the lysosomes. The term autophagy refers to the collection of various cellular processes including macroautophagy, microautophagy, chaperone-mediated autophagy and non-canonical autophagy. Macroautophagy is the major route for degradation of cytoplasmic constituents where cellular components are sequestered within a double-membrane structure called autophagosome, followed by its fusion with lysosomes. Autophagy is a tightly regulated phenomenon and its dysregulation results in various diseases. It has been reported that autophagy can

Veterinary Microbiology, Aug 1, 1994

We have characterised two groups of adenoviruses isolated from sheep in Australia. Restriction en... more We have characterised two groups of adenoviruses isolated from sheep in Australia. Restriction endonuclease maps for enzymes BamHI, ClaI, SalI, SmaI and SphI have been determined for the genome of ovine adenoviruses related to bovine adenovirus serotype 7 (BAV 7) from sheep in Western Australia. Although previously serotyped as BAV 7 these isolates are different from bovine isolates of BAV 7 based on comparison with published restriction endonuclease profiles and maps of BAV 7 cattle isolates. Additional adenovirus isolates obtained from Victorian sheep have been serotyped as ovine adenovirus type 5 (OAV 5). On the basis of restriction endonuclease analysis these viruses are different from the sheep BAV 7 isolates. Following infection of sheep with ovine BAV 7 and OAV 5 isolates, virus was recovered from nasal and rectal swabs for several days. Antibodies detected by ELISA and serum neutralisation tests (SN) developed by 15 days after infection. Virus also spread from the infected sheep to an incontact control and one of ten sheep purchased for infection studies had SN antibodies to BAV 7 suggesting that BAV 7-like viruses naturally infect sheep in Victoria and Western Australia. With further development, these ovine adenoviruses may be suitable as vectors for the delivery of vaccine antigens to sheep and cattle.

Journal of General Virology, May 1, 2017

Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection... more Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection. Recent studies have shown that Japanese encephalitis virus (JEV)-induced autophagy negatively influences virus replication in mouse neuronal cells and embryonic fibroblasts, and delays virus-induced cell death. Here, we evaluated the role of ER stress pathways in inducing autophagy during JEV infection. We observed that JEV infection of neuronal cells led to activation of all three sensors of ER stress mediated by eIF2a/PERK, IRE1/XBP1 and ATF6. The kinetics of autophagy induction as monitored by levels of SQSTM1 and LC3-II paralleled activation of ER stress. Inhibition of the eIF2a/PERK pathway by siRNA-mediated depletion of proteins and by the PERK inhibitor had no effect on autophagy and JEV replication. However, depletion of XBP1 and ATF6, alone or in combination, prevented autophagy induction and significantly enhanced JEV-induced cell death. JEVinfected cells depleted of XBP1 or ATF6 showed reduced transcription of ER chaperones, ERAD components and autophagy genes, resulting in reduced protein levels of the crucial autophagy effectors ATG3 and BECLIN-1. Conversely, pharmacological induction of ER stress in JEV-infected cells further enhanced autophagy and reduced virus titres. Our study thus demonstrates that a crucial link exists between the ER stress pathways and autophagy in virus-infected cells, and that these processes are highly regulated during virus infection.

bioRxiv (Cold Spring Harbor Laboratory), Jan 18, 2022

Disease Models & Mechanisms, Oct 1, 2021

Singh, ATPC, Regional Centre for Biotechnology, Faridabad, India, for his scientific and technica... more Singh, ATPC, Regional Centre for Biotechnology, Faridabad, India, for his scientific and technical inputs during the mass spectrometry experiments and data analysis. We are also Disease Models & Mechanisms • DMM • Accepted manuscript thankful to Mr. Nagavara Prasad G. and Mr. Subodh Jain for their technical assistance during mass spectrometry experiments.

MSystems, Dec 17, 2019

Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein ... more 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 Ϫ/Ϫ 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 ␤ (TGF-␤) 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 Ϫ/Ϫ 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 doublestranded 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 autophagydeficient 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.

Current Science, Feb 10, 2010

1National Brain Research Centre, Manesar 122 050, India 2Department of Biochemistry, Indian Insti... more 1National Brain Research Centre, Manesar 122 050, India 2Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India 3National Institute of Immunology and Vaccine and Infectious Disease Research Center, THSTI, New Delhi 110 067, India

Journal of Virology, Mar 1, 1996

Previously we identified the locations of three neutralization epitopes (a, b1, and b2) of Ross R... more Previously we identified the locations of three neutralization epitopes (a, b1, and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C. A. Fernon, L. Dalgarno, and R. C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no differences, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four-to fivefold-higher 50% lethal dose (LD 50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The LD 50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.

Journal of Antimicrobial Chemotherapy, Nov 23, 2011

Background: Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in SouthEast... more Background: Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in SouthEast Asia and there is a pressing need to develop novel therapeutic options against it. Methods: Gene silencing by RNA interference has therapeutic potential by way of degrading the RNA genome of JEV. Four small hairpin RNAs (shRNAs) targeting different locations in the JEV genome were evaluated for antiviral activity against JEV in different cell lines and the mouse model of disease. Results: shN8010, an shRNA targeting the NS5-coding sequence of JEV, had significant antiviral activity in cultured cells. JEV titres were suppressed by 99% in human embryonic kidney cells at 24 h post-infection (p.i.) when shN8010 was delivered using a plasmid. Further, shN8010 delivered using recombinant adenovirus caused 99% and 95% suppression of JEV titres at 24 h p.i. in porcine stable kidney and Vero cells, respectively. In Neuro-2a cells, JEV titres at 24 h p.i. were suppressed by 90% when shN8010 was delivered using a recombinant adenovirus or retrovirus. Four-week-old FvB/J mice treated intracerebrally with recombinant adenovirusdelivered shN8010 1 week prior to lethal intraperitoneal JEV challenge showed no protection, although the mean survival time was prolonged. In a similar experiment, retrovirus-delivered shN8010 provided 100% protection to mice following the lethal JEV challenge. Conclusions: NS5-targeting shRNA (shN8010) had very significant antiviral activity in both cultured cells and the mouse model of JEV infection.

Molecular Aspects of Medicine, Oct 1, 2021

Japanese encephalitis virus (JEV) is a flavivirus, spread by the bite of carrier Culex mosquitoes... more 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.

Journal of Virology, Feb 1, 2015

The beginning of the second century of research in the field of virology (the first virus was dis... more The beginning of the second century of research in the field of virology (the first virus was discovered in 1898) was marked by its amalgamation with bioinformatics, resulting in the birth of a new domain-viroinformatics. The availability of more than 100 Web servers and databases embracing all or specific viruses (for example, dengue virus, influenza virus, hepatitis virus, human immunodeficiency virus [HIV], hemorrhagic fever virus [HFV], human papillomavirus [HPV], West Nile virus, etc.) as well as distinct applications (comparative/diversity analysis, viral recombination, small interfering RNA [siRNA]/short hairpin RNA [shRNA]/microRNA [miRNA] studies, RNA folding, protein-protein interaction, structural analysis, and phylotyping and genotyping) will definitely aid the development of effective drugs and vaccines. However, information about their access and utility is not available at any single source or on any single platform. Therefore, a compendium of various computational tools and resources dedicated specifically to virology is presented in this article.

Protein Expression and Purification, Mar 1, 2003

The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like part... more The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system. We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli. The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E. coli. The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure. The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides. This resulted in a single amino acid change at the N-terminus of the protein. The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E. coli. The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E. coli to form VLPs.