Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis

Abstract

Neurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells.

Fig 1. Comparison of human neuroblastoma proteome modulation during JEV or WNV infection.

A-B. Volcano plots of protein expression in non-infected vs infected cells. SK-N-SH cells were infected for 48h with either JEV (A) or WNV (B) and proteins were extracted for label free quantitation by mass spectrometry. The results are representative of 3 independent experiments. Each spot represents a protein identified (black) and significantly down-regulated (green) or up-regulated (red) during viral infection. C. Venn diagram of the proteins up- and down-regulated in JEV (A) and WNV (B) infected cells.

Fig 2. Functional clustering and network analysis of proteins modulated during JEV infection.

A. Percentage of proteins up(red)- and down(green)-regulated in functional groups. Modulated proteins were clustered into functional groups using DAVID v6.8 (detailed in S1 Fig). Functional groups are organized in four domains: Biological process, Molecular function, Cellular component or Structural feature and according to their enrichment score from the left to the right. Total number of proteins associated to each group is noted between brackets. B. Networks of up(red)- and down(green)-regulated proteins. Protein-protein interactions (PPI) networks were determined with STRING v10 and visualized with Cytoscape. Proteins regulated in common with WNV are highlighted in bold. Node size is relative to the number of edges. Grey nodes correspond to second shell proteins linking identified proteins. Edges are determined according to the number of sources (text mining, experiments, databases or co-expression) supporting the link between proteins.

Fig 3. Functional clustering and network analysis of proteins modulated during WNV infection.

A. Percentage of proteins up(red)- and down(green)-regulated in functional groups. Modulated proteins were clustered into functional groups using DAVID (detailed in S2 Table). Functional groups are organized in four domains: Biological process, Molecular function, Cellular component or Structural feature and according to their enrichment score from left to right. Total number of proteins associated to each group is noted between brackets. B. Networks of up(red)- and down(green)-regulated proteins. PPI networks were determined with STRING and visualized with Cytoscape. Proteins regulated in common with JEV are highlighted in bold. Node size is relative to the number of edges. Grey nodes correspond to second shell proteins linking identified proteins. Edges are determined according to the number of sources (text mining, experiments, databases or co-expression) supporting the link between proteins.

Fig 4. JEV and WNV inhibit collagen, PAM and SSBP2 expression in a neuron model.

SK-N-SH cells were infected with JEV or WNV at a MOI of 1 and proteins were extracted for western blot analysis 48hpi. Two independent experiments are displayed on the blot.

Fig 5. Comparison of modulation of the human neuroblastoma proteome during JEV or WNV infection in the presence of mosquito SGE.

Volcano plots of protein expression in non-infected vs infected cells. After 48h, proteins from mock (A), JEV (B) or WNV (C) infected SK-N-SH cells were extracted for label free quantification by mass spectrometry. The results are representative of 3 independent experiments. Each spot represents a protein identified (black) and significantly down-regulated (blue) or up-regulated (red) during viral infection.

Fig 6. Functional clustering analysis of proteins modulated by mosquito SGE during JEV or WNV infection.

Percentage of proteins up(red)- and down(green)-regulated in functional groups after JEV (A) or WNV (B) infection in the presence of mosquito SGE. Modulated proteins were clustered into functional groups using DAVID. Functional groups are organized in four domains: Biological process, Molecular function, Cellular component or Structural feature and according to their enrichment score from left to right. Total number of proteins associated to each group is noted between brackets.