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MS-based proteomics of Adeonvirus type 2 infected primary human cells

ABSTRACT: To combat virus infections, which are major human killers, a deeper understanding of how viruses reprogram their hosts to create optimal production of progeny is needed. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from studies of mRNA expression. Here, we investigated the changes in cellular protein expression during the late phase of adenovirus type 2 (Ad2) infection of primary human cells by stable isotope labeling in cell culture (SILAC) with subsequent liquid chromatography-high resolution tandem mass spectrometric (LC-MS/MS) analysis using a Q-Exactive Orbitrap instrument. Cells were in-depth evaluated 24 and 36 hours post infection (hpi) and two biological replicates were investigated using swapped labeling. In total, 2648 and 2394 proteins were quantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated more than 1.6-fold at the two time points, and approximately 80% of them had sustained regulation from 24 to 36 hpi. For the deregulated genes the change in protein expression was compared with that of RNA expression using cDNA sequencing data from the same time points of infection. The comparison revealed a surprisingly low correlation (r = 0.3). Consistency between changes in RNA and protein expression was observed for approximately 30% of the genes, whereas a majority showed either divergent or opposite expression profiles. The deregulated proteins were subjected to pathway and biological function analysis using bioinformatics tools. A surprising observation was that proteins related to carbohydrate metabolism were up-regulated at the protein level but unchanged at the RNA level whereas histone proteins were down-regulated at the protein level but up-regulated at the RNA level. Our results show that regulation of cellular gene expression in the response to viral infection is more complex than hitherto believed and several examples of posttranscriptional regulation of gene expression were observed. The results contribute to our understanding of host cell gene regulation during infection at a deeper level.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Human Adenovirus C Serotype 2 (hadv-2) (human Adenovirus 2) Homo Sapiens (human)

TISSUE(S): Fibroblast Of Lung, Cell Culture

DISEASE(S): Adenovirus Infectious Disease

SUBMITTER: Sara Lind

LAB HEAD: Sara Lind

PROVIDER: PXD004095 | Pride | 2016-12-20

REPOSITORIES: Pride

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			Action	DRS
	20140312_Sara_SILAC_01.raw	Raw
	20140312_Sara_SILAC_02.raw	Raw
	20140312_Sara_SILAC_03.raw	Raw
	20140312_Sara_SILAC_04.raw	Raw
	20140312_Sara_SILAC_05.raw	Raw

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Project description:A first generation Affymetrix GeneChipÂ® Porcine genome array was used to profile the gene expression in porcine mesenteric lymph nodes over a time course of infection with S. Typhimurium, including the acute (8 hours post inoculation (hpi), 24 hpi, 48 hpi) and chronic (21 days post-inoculation (dpi)) stages of infection. Our objectives were to 1) identify and examine the stereotypical gene expression response within host MLN to S. Typhimurium infection, 2) characterize global host responses by revealing the specific features of the hostâs innate immunity pathways, and 3) explore if and how S. Typhimurium may escape the host immune response and develop into a carrier state. Our study has attempted to investigate the features of host gene expression profiling during S. Typhimurium infection at the acute and chronic infection stages and to explore the mechanism by which S. Typhimurium can escape from the host immune response and develop a carrier state in the host. In conclusion, by using the Affymetrix porcine GeneChip, 848 differentially expressed genes were identified in porcine MLN during infection and several specific features of host response were revealed by gene cluster and pathway analysis. Our data are the first report to investigate global host responses to S. Typhimurium in porcine MLN, and this new study provides data applicable for studying enteric salmonellosis of pigs, as well as humans. Experiment Overall Design: Fifteen piglets from Salmonella spp.-free sows were weaned at 10 days (d) of age, shipped to the National Animal Disease Center, Ames, IA and raised in isolation facilities. To confirm that all piglets were free of Salmonella spp. prior to challenge, bacteriological cultures were performed on rectal swabs twice. Seven week old pigs were randomly divided into 2 groups, 3 non-infected pigs and 12 infected pigs. Three non-infected control pigs were necropsied 2 days prior to experimental infection. On day 0, pigs in the infected groups were intranasally challenged with 1 billion CFU of Salmonella enterica serovar Typhimurium. Three infected pigs were necropsied at 8 hours post-inoculation (hpi), 24 hpi, 48 hpi and 21 days post-inoculation (dpi). Tissue samples from the mesenteric lymph nodes (MLN) were collected and immediately frozen in liquid nitrogen for RNA isolation.

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MS-based proteomics of Adeonvirus type 2 infected primary human cells

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