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

Mechanisms of Loss of Functions of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

Background: Mutations in the coding region of angiogenin (ANG) gene have been found in patients suffering from Amyotrophic Lateral Sclerosis (ALS). Neurodegeneration results from the loss of angiogenic ability of ANG (protein coded by ANG). In this work, we performed extensive molecular dynamics (MD) simulations of wild-type ANG and disease associated ANG variants to elucidate the mechanism behind the loss of ribonucleolytic activity and nuclear translocation activity, functions needed for angiogenesis. Methodology/Principal Findings: MD simulations were carried out to study the structural and dynamic differences in the catalytic site and nuclear localization signal residues between WT-ANG (Wild-type ANG) and six mutants. Variants K17I, S28N, P112L and V113I have confirmed association with ALS, while T195C and A238G single nucleotide polymorphisms (SNPs) encoding L35P and K60E mutants respectively, have not been associated with ALS. Our results show that loss of ribonucleolytic activity in K17I is caused by conformational switching of the catalytic residue His114 by 99u. The loss of nuclear translocation activity of S28N and P112L is caused by changes in the folding of the residues 31 RRR 33 that result in the reduction in solvent accessible surface area (SASA). Consequently, we predict that V113I will exhibit loss of angiogenic properties by loss of nuclear translocation activity and L35P by loss of both ribonucleolytic activity and nuclear translocation activity. No functional loss was inferred for K60E. The MD simulation results were supported by hydrogen bond interactio

Relative fold change in differentially expressed proteins in brain tissue post-JEV infection.

<p>Spot intensities were normalized by total valid spot intensities and mean of values from duplicate analytical gels from four biological replicates were subjected to paired <i>t</i>-test analysis. Protein spots showing altered expression between control and experimental groups (|ratio|≥1.5, <i>p</i><0.05) were marked and excised. Dark grey bars indicate spots from mock infected control sample while light grey bars indicate JEV infected sample. * = p<0.05, # = p<0.01. Data represented are means ± SD of four independent experiments.</p

Comparison of AutoDock and ParDOCK binding energy scores for NCI-65828 complexes with WT-ANG and ANG mutants.

<p>*(ANG in which there is no conformational change of the catalytic residue His114).</p

Confirmation of JEV infection in both <i>in vivo</i> and <i>in vitro</i> models using JEV specific antibody.

<p>The onset of JEV infection was confirmed by immunostaining of mouse brain sections and Neuro2a cells using JEV specific antibodies.</p

Expression of selected proteins in human neuroblastoma (SHSY 5Y) cells after JEV infection.

<p>SHSY 5Y cells were either mock-infected or infected with JEV (4.4×10⁹ pfu/mL) at multiplicity of infection (MOI) of 5 for 1.5 h. The expression of different proteins after JEV infection as compared to mock-infected controls was analyzed by immunoblotting. Data represented are means ± SD of three independent experiments. Dark grey bars indicate spots from mock infected control sample while light grey bars indicate JEV infected sample * = p<0.05.</p

Differential protein expression in JEV infected Neuro2A cells.

<p>(<b>A</b>) A representative 2-DE gel of Neuroblastoma cell (Neuro2a) lysate from mock-infected and JEV infected cells. Equal amount of total protein from cell lysates were separated on an immobilized linear pH gradient IPG strips (4.0–7.0) and then in the second dimension on 10% SDS-PAGE. Spots showing differential expression were marked excised, and identified by MALDITOF/MS and database searches. The spots are labeled on the gel according to the numbers presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090211#pone.0090211.s007" target="_blank">table S1</a>. (<b>B</b>) Protein spots representing relative fold change in expression in mouse neuroblastoma cell (Neuro2a) lysate.Spot intensities were normalized by total valid spot intensities and mean of values from duplicate analytical gels from four biological replicates were subjected to paired <i>t</i>-test analysis. Protein spots showing altered expression between control and experimental groups (|ratio|≥1.5, <i>p</i><0.05) were marked and excised. Dark grey bars indicate spots from mock infected control sample while light grey bars indicate JEV infected sample. * = p<0.05. Data represented are means ± SD of four independent experiments.</p

Validation of proteomic results using Immunostaining.

<p>Differential expression of selected proteins in neuron following JEV-infection in <i>in vivo</i> model. Images are representative of three independent experiments.</p