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Analysis of the differential host cell nuclear proteome induced by attenuated and virulent hemorrhagic arenavirus infection.

ABSTRACT: Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host response is a potential strategy for the treatment of infectious diseases. We have previously investigated the global host response to attenuated and lethal arenavirus infections by using high-throughput immunoblotting and kinomics approaches. In this report, we describe the differential nuclear proteomes of a murine cell line induced by mock infection and infection with attenuated and lethal variants of PICV, investigated by using two-dimensional gel electrophoresis. Spot identification using tandem mass spectrometry revealed the involvement of a number of proteins that regulate inflammation via potential modulation of NF-kappaB activity and of several heterogeneous nuclear ribonuclear proteins. Pathway analysis revealed a potential role for transcription factor XBP-1, a transcription factor involved in major histocompatibility complex II (MHC-II) expression; differential DNA-binding activity was revealed by electrophoretic mobility shift assay, and differences in surface MHC-II expression were seen following PICV infection. These data are consistent with the results of several previous studies and highlight potential differences between transcriptional and translational regulation. This study provides a number of differentially expressed targets for further research and suggests that key events in pathogenesis may be established early in infection.

SUBMITTER: Bowick GC

PROVIDER: S-EPMC2612403 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Analysis of the differential host cell nuclear proteome induced by attenuated and virulent hemorrhagic arenavirus infection.

Bowick Gavin C GC Spratt Heidi M HM Hogg Alison E AE Endsley Janice J JJ Wiktorowicz John E JE Kurosky Alexander A Luxon Bruce A BA Gorenstein David G DG Herzog Norbert K NK

Journal of virology 20081112 2

Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host ...[more]

PMID: 19004951

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Project description:Lassa fever virus is a zoonotic pathogen that plagues the endemic areas of West Africa. Rhesus macaques infected with a related arenavirus, LCMV-WE, serve as a model for Lassa-infection of human beings. Using a dose similar to that expected from a needle-stick, monkeys experience an early pre-viremic phase (day 1-3), a viremic phase with febrile onset (day 4-7), and, like human beings who succumb, they die within two weeks. Our goal was to monitor changes in gene expression that parallel disease progression in an effort to 1) identify genes with altered expression after infection, 2) identify genes that could discriminate between a virulent and non-virulent infection, and 3) identify genes encoding products that could serve as treatment targets for FDA-approved pharmaceuticals. Genes related to all three of these categories have been identified and some have been given preliminary validation by quantitative PCR and proteomic studies. These genes will be valuable candidates for future validation as prognostic biomarkers; We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: Rhesus macaques inoculated with a lethal dose of LCMV-WE were used to model human infection with Lassa fever virus (Lukashevich et al 2002, 2003; Rodas et al 2004). To identify molecular events that occur during the first few days of virus exposure, we monitored gene expression in peripheral blood mononuclear cell (PBMC) samples by microarray analysis. Approximately 150 samples from 23 different rhesus macaques (Table 1) were hybridized to Affymetrix U133 chips. Experiment Overall Design: Affymetrix U133 arrays cover the entire human genome with approximately 54,000 gene fragments representing approximately 22,000 genes. The use of these human arrays for macaque transcriptome analysis has been validated by other laboratories (Ace et al, 2004 Wang et al, 2004), i.e. 99.8% of the array elements for which human DNA gave a signal >1.4-fold over background were also >1.4 over background for macaque DNA (Rubins et al, 2004). LCMV-infected blood samples were compared to uninfected controls and genes with over two-fold change in expression were designated âdifferentially expressedâ (Fig. 1). Thus, out of 54,000 elements, 8410 (16%) were differentially regulated with a >2-fold change in expression [supplementary Table 1 has these genes in order of the magnitude of their differential expression]. Experiment Overall Design: Virus was detectable in these monkeys at day 4 after infection by plaque assay and by RT-PCR of blood cDNA (Djavani et al 2005). Although virus is replicating in monocytes, endothelial cells, liver, spleen, and peripheral lymphoid organs soon after inoculation (Rai et al, 1997; Lukashevich et al, 2002), virus is not detectable in the circulation for the first three days. Thus we define the early, pre-viremic stage as days 1-3, and a later viremic stage as days 4-7. Although the LCMV-Armstrong strain replicated as well in cell culture as LCMV-WE, the LCMV-Arm-infected monkeys did not experience viremia, i.e. viral nucleic acid were detected in tissues but virus loads were not high enough to be detected in the circulation. The Arm-infected monkeys resisted a lethal challenge with LCMV-WE, further confirming that they had been productively infected (Rodas et al, 2004). An overview of gene expression in monkeys shows a trend to down-regulate genes at the onset of viremia followed by a sharp increase in up-regulated genes. Those monkeys that did not experience viremia (Armstrong-infected) showed no dramatic down-regulation trend (Fig. 1). Over the entire study period of 7 days, the number of down-regulated genes in the WE-infected macaques was significantly more than the number seen in the Arm-infected monkeys (p < 0.005).

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Analysis of the differential host cell nuclear proteome induced by attenuated and virulent hemorrhagic arenavirus infection.

Publications

Analysis of the differential host cell nuclear proteome induced by attenuated and virulent hemorrhagic arenavirus infection.

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