Proteomics analysis of differential expression of cellular proteins in response to avian H9N2 virus infection in human cells

Abstract

We present the first proteomic analysis on the cellular responses to avian influenza virus (H9N2) infection in a human cell line in different time courses in order to search for target proteins for viral pathogenesis/adaptation studies. By using 2-DE coupled with MALDI-TOF MS and nano-ESI-MS/MS, we identified a set of differentially expressed cellular proteins, including cytoplasmic actin, cytokeratin, prohibitin, enoyl-CoA hydratase, peptide-prolyl cis-trans isomerase A (PPIase A), chloride intracellular channel protein 1, pyruvate dehydrogenase E1 component subunit beta, adenine phosphoribosyltransferase, guanine nucleotide-binding protein subunit beta, nucleoside diphosphate kinase A, elongation factor 1-beta and splicing factor, arginine/serine rich 1. The most significant changes in different time courses were found in cytoplasmic actin and cytokeratin, both of which constituted the major components of cytoskeleton network in the cells. The obtained data suggested a possible role of the cytoskeleton during avian influenza virus infection of mammalian cells, which might help for better understanding of the dynamics of avian influenza virus and host interaction in mammalian cell setting.

Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells

“…While 2-D DIGE is excellent for resolving protein species that differ in posttranslational modification, such as phosphorylation, it suffers several drawbacks, including a relatively low dynamic range and sample overloading (13), variability in labeling efficiency, as well as labeling deficits for proteins lacking lysine or cysteine residues, and is unsuited for proteins at the extremes of molecular weight, alkalinity, or hydrophobicity (59). Finally, in-gel digestion methods are usually less efficient in allowing peptide identification than in-solution digestion, which may partially explain why earlier studies identified less than 25 differentially regulated proteins (48,72).…”

“…Because viral infection leads to both qualitative and quantitative effects on host gene expression and function, we have complemented these previous studies by deriving a quantitative proteomic assessment of influenza infection to further define the effects of influenza virus infection on host functions. Whereas a variety of quantitative proteomic methods have been employed to examine perturbations in host protein quantities after virus infection, quantification of host protein responses after influenza virus infection had only previously been reported after 2-D DIGE analysis, which identified 25 or fewer proteins (48,72). Here, we present the application of SILAC and demonstrate several advantages relative to this earlier approach.…”

“…Thus, proteomic analyses have also been employed to better understand host alterations to virus infection. Vester et al used two-dimensional difference in gel electrophoresis (2-D DIGE) and identified 8 significantly altered host proteins in influenza virus A/PR/8/34 (H1N1)-infected MDCK and human A549 cells (72), and Liu and colleagues used a similar approach to identify about 25 significantly altered host proteins in avian influenza A/Hong Kong/108/2003 (H9N2)-infected human gastric carcinoma cells (48).…”

Macaque Proteome Response to Highly Pathogenic Avian Influenza and 1918 Reassortant Influenza Virus Infections

“…From the limited number of previous proteomic investigations of influenza virus-host protein expression (1,30,52), the most relevant comparison of results for the current study involves the previous in-depth LC-MS/MS analysis of Baas et al (1). To update the Baas et al (1) data set analysis for this comparison, we reanalyzed the original spectra from the study against the macaque protein database, as the original data comparison was performed using a human protein list, which resulted in an increase in the number of peptide identifications from 14,100 to 15,322.…”

“…Previous work studying the host proteome response to influenza virus infection has primarily targeted in vitro systems, specifically, human cell lines (30,35,52). Vester et al and Liu et al both utilized global two-dimensional PAGE (2D-PAGE) separation approaches and reported 16 and 22 differentially abundant proteins, respectively.…”