Quantitative Whole-Cell Proteome Analysis of Pseudorabies Virus-Infected Cells

Abstract

A quantitative proteome study using the stable isotope labeling with amino acids in cell culture technique was performed on bovine kidney cells after infection with the alphaherpesvirus pseudorabies virus (PrV), the etiological agent of Aujeszky's disease. To enhance yields of proteins to be identified, raw extracts were fractionated by affinity solid-phase extraction with a combination of a cibacron blue F3G-A and a heparin matrix and with a phosphoprotein-specific matrix. After two-dimensional gel electrophoresis in different pH ranges between pH 3 and pH 10, 2,600 proteins representing 565 genes were identified by mass spectrometry and screened for virus-induced changes in relative protein levels. Four hours after infection, significant quantitative variations were found for constituents of the nuclear lamina, representatives of the heterogeneous nuclear ribonucleoproteins, proteins involved in membrane trafficking and intracellular transport, a ribosomal protein, and heat shock protein 27. Several proteins were present in multiple charge variants that were differentially affected by infection with PrV. As a common pattern for all these proteins, a mass shift in favor of the more acidic isoforms was observed, suggesting the involvement of viral or cellular kinases.Herpesviral genes are generally expressed in three kinetic classes (18,19), which are regulated sequentially by a number of positive and negative feedback mechanisms exerted by virusencoded proteins. However, herpesvirus infection also influences the expression of cellular genes, e.g., by host cell shutoff mechanisms that target the integrity of cellular mRNA and thus block the synthesis of cellular proteins. Herpes simplex virus type 1 (HSV-1), the prototypical alphaherpesvirus, expresses two shutoff proteins, ICP27 and pUL41. Whereas ICP27 interferes with mRNA splicing (16, 17), pUL41 degrades mRNA by virtue of its endoribonucleolytic activity (10, 29, 54), with specificity for mRNAs containing AU-rich elements (11). Homologs of both proteins are also present in pseudorabies virus (PrV), an alphaherpesvirus causing Aujeszky's disease. PrV, whose main host is the pig, infects numerous mammalian species except higher primates including humans. In contrast to HSV-1 ICP27, the PrV homolog pUL54 is dispensable for virus replication in cell culture (47,49). Transcript analyses of PrV-infected rat (44), human (6), and porcine (12) cells demonstrated alterations in the abundance of individual cellular transcripts, which resulted in the depletion or accumulation of specific mRNAs. Of the 9,850 genes examined in one study (6), the number of significantly up-or downregulated genes increased from approximately 1,000 to over 2,400 between 6 and 9 h after infection. Evaluation of the functions annotated for highly regulated cellular genes shows that PrV infection influences numerous cellular pathways and that genes involved in protein and nucleic acid metabolism, signaling, transport, cell cycle control, adhesion, transcription, the stress response, and innate ...

Histone Deacetylases in Herpesvirus Replication and Virus-Stimulated Host Defense

“…Quantitative mass spectrometry incorporating metabolic labeling has been successfully used to assess changes in protein expression levels during pseudorabies virus (PRV) infection, identifying alterations in expression patterns for proteins involved in intracellular transport, translation and host stress response [118]. Similarly, identification of dynamic host-virus protein interactions during infection has contributed significantly to the understanding of both virus-mediated effects on host cell functions and of host defense mechanisms in response to infection (e.g., [38,119,120,121,122,123,124,125,126]).…”

Isolation and Preliminary Characterization of Herpes Simplex Virus 1 Primary Enveloped Virions from the Perinuclear Space

“…There have been extensive proteomics studies for various members of the herpesvirus family, such as HCMV, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, among others (1,14,42). However, it was not until recently that these types of largescale proteomic analyses were carried out for PrV and HSV-1 (19,37). The aim of this study was to isolate the HSV-1 primary enveloped virion and perform whole-particle proteomic analysis to elucidate its components.…”

Influence of insertion site of the avian influenza virus haemagglutinin (HA) gene within the Newcastle disease virus genome on HA expression

“…This procedure is highly precise and has been used for the quantification of cellular proteins in proteome studies but also to determine expression levels of viral proteins (Skiba et al, 2008(Skiba et al, , 2010. In preceding 2D electrophoretic analyses with cell extracts from uninfected and NDV-infected cells and cells infected with one of the recombinants, HA, the NDV-specific P and a number of cellular proteins were located in 2D gels and identified by mass spectrometry and in 2D Western blots.…”

“…Relative HA protein expression levels in the NDV/AIV mutants were assayed by a combination of two-dimensional (2D) electrophoresis and quantitative mass spectrometry using the stable isotope labelling with amino acids in cell culture (SILAC) technique (Ong et al, 2002) with some modifications (Skiba et al, 2008). This procedure is highly precise and has been used for the quantification of cellular proteins in proteome studies but also to determine expression levels of viral proteins (Skiba et al, 2008(Skiba et al, , 2010.…”