Proteomic Analysis of Host Responses in HepG2 Cells during Dengue Virus Infection

Abstract

Dengue virus infection remains a public health problem worldwide. However, its pathogenic mechanisms and pathophysiology are still poorly understood. We performed proteomic analysis to evaluate early host responses (as indicated by altered proteins) in human target cells during dengue virus infection. HepG2 cells were infected with dengue virus serotype 2 (DEN-2) at multiplicity of infection (MOI) of 0.1, 0.5, and 1.0. Quantitative analyses of DEN-2 infection and cell death at 12, 24, and 48 h postinfection showed that the MOI of 1.0 with 24 h postinfection duration was the optimal condition to evaluate early host responses, as this condition provided the high %Infection ( approximately 80%), while %Cell death ( approximately 20%) was comparable to that of the mock-control cells. Proteins derived from the mock-control and DEN-2-infected cells were resolved by 2-D PAGE ( n = 5 gels for each group) and visualized by SYPRO Ruby stain. Quantitative intensity analysis revealed 17 differentially expressed proteins, which were successfully identified by peptide mass fingerprinting. Most of these altered proteins were the key factors involved in transcription and translation processes. Further functional study on these altered proteins may lead to better understanding of the pathogenic mechanisms and host responses to dengue virus infection, and also to the identification of new therapeutic targets for dengue virus infection.

Dengue Virus Infection Induces Upregulation of GRP78, Which Acts To Chaperone Viral Antigen Production

“…Of these seven proteins, altered protein expression was confirmed by Western analysis for two proteins; elongation factor Tu (EF-Tu) mitochondrial precursor and vinculin. Altered GRP78 protein levels were not identified in this study (32). Using 2DGE and MS analysis, we successfully identified GRP78 as significantly upregulated in DENV-infected cells.…”

“…The total number of spots detected was low and could be improved technically by cup loading rather than passive rehydration of IEF strips, use of a more sensitive protein stain, and fractionation of cell lysates to reduce protein complexity. A proteomic study of DENV-infected HepG2 cells identified 800 protein spots and 17 differentially expressed proteins, only 7 of which were altered by Ͼ2-fold, the cutoff used in our proteomic study (32). Of these seven proteins, altered protein expression was confirmed by Western analysis for two proteins; elongation factor Tu (EF-Tu) mitochondrial precursor and vinculin.…”

“…Transcriptome analyses of DENV-infected endothelial cells (23,45), HepG2 cells (9), circulating patient cells (39), or peripheral blood mononuclear cells from DENV-infected macaque monkeys (37) have identified alterations in transcripts involved in a variety of cellular processes, including the innate immune response, cell signaling, and metabolic processes. A recent study examined proteomic changes in DENVinfected HepG2 cells and identified 17 altered cellular proteins, including 2 proteins for which the changes were confirmed (32). In the current study, we performed a proteomic analysis of changes due to DENV infection in a target cell population that is relevant to a natural DENV infection and in which DENV-induced cytopathic effect (CPE) and the cellular death response are absent.…”

Proteomic Analysis of Interactions between a Deep-Sea Thermophilic Bacteriophage and Its Host at High Temperature

“…Upregulation of the chaperonin GroEL in the host could be due to the presence of misfolded or aggregated proteins under stress conditions. The elongation factors EF-Tu and EF-Ts have been found to bind tightly to the viral RNAdependent RNA polymerase and have a major function in the delivery of aminoacyl-tRNA (aa-tRNA) to the A site on the ribosome during the translation of mRNA into protein (18). In our study, the upregulation of EF-Ts in the GVE2-infected Geobacillus sp.…”

“…Proteomics approaches have become increasingly popular in studies to reveal the effects of viral infection on the cellular proteome (1,27). Comparative proteomic approaches coupling two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) are widely used to analyze host responses in animals, humans, and plants during virus infection (18,19,20). The approaches effectively facilitate investigations of the molecular profiles of virus-infected cells (27).…”