Quantification of the Host Response Proteome after Mammalian Reovirus T1L Infection

<div><p>All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular “transcriptome.” We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.</p> </div

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

Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular “transcriptome.” Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules. We have used stable isotope labeling by amino acids in cell culture (SILAC), combined with high-throughput two-dimensional (2-D) high-performance liquid chromatography (HPLC)/mass spectrometry, to determine quantitative differences in host proteins after infection of human lung A549 cells with human influenza virus A/PR/8/34 (H1N1) for 24 h. Of the 4,689 identified and measured cytosolic protein pairs, 127 were significantly upregulated at >95% confidence, 153 were significantly downregulated at >95% confidence, and a total of 87 proteins were upregulated or downregulated more than 5-fold at >99% confidence. Gene ontology and pathway analyses indicated differentially regulated proteins and included those involved in host cell immunity and antigen presentation, cell adhesion, metabolism, protein function, signal transduction, and transcription pathways

Kinetics of reovirus growth and viral-induced cytopathology.

<p>Each of five different cell lines (L929, A549, HEK293, CaCo₂ and Hela) were infected at MOI  = 1 PFU/cell with T1L (a) or T3D (b). Cell lysates were harvested at 0, 24, 48 and 72hpi and titrated. Experiments were performed in triplicate; error bars represent standard error. Virus titers were greatest in the L929 and HEK293 cells for both virus strains. HEK293 (c) and L929 (d) cells were then re-analyzed as in (a) and (b) after infection at MOI  = 5 and at additional time points. Aliquots of the infections in (c) and (d) were also assessed for cell viability by trypan blue exclusion (e and f, respectively), with 100 μg/ml puromycin used as a positive cell killing control. Experiments were performed in duplicate; error bars represent standard error.</p

HEK293 proteins increased >95% confidence^a.

a<p>Protein is included if at least half of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates.</p>b<p>L/H ratio refers to the geometric mean of all log₂ L/H values for each given gi number, expressed as relative protein quantity in infected cultures.</p

Top network functions generated using Ingenuity protein analysis for HEK293 cells infected with T1L reovirus at (a) 6hpi and (b) 24hpi.

<p>Graphs represent host cell functions with highest score (x-axis) based on the number of differentially regulated proteins observed in that network. The higher the score, the greater the number of proteins differentially regulated in that particular function network.</p

HEK293 proteins decreased >95% confidence^a.

a<p>Protein is included if at least half of the biologic z-score values are ≥1.960σ (indicated by bolding) and there are no major disagreements between biological replicates.</p>b<p>L/H ratio refers to the geometric mean of all log₂ L/H values for each given gi number, expressed as relative protein quantity in infected cultures.</p

Canonical pathway “Activation of IRF by cytosolic pattern recognition receptors” that was identified as significantly (p-value of 3.7×10⁻⁵) altered after 24 h of T1L reovirus infection in HEK293 cells.

<p>Protein regulation expression patterns overlaid from first biological replicate. Red indicates up-regulation, grey represents proteins not changed in abundance after infection and white represents molecules not identified in SILAC experiment but are part of the known canonical pathway.</p

Identified protein analysis.

<p>Venn diagram summaries of the three separate T1L reovirus SILAC experiments at (a) 6hpi, when a total of 3076 unique proteins were identified and at (b) 24hpi where a total of 2992 unique proteins were identified. Overlapping numbers represent those proteins identified in more than one biological replicate (c) Population distribution represented by the log₂ of L:H ratios of identified proteins plotted against the protein counts. This population distribution is used to determine the z-scores indicating those proteins considered significantly up or down regulated. Most proteins are seen at a 1∶1 ratio after infection, indicated by the peak of the population distribution indicated at 0. For clarity, only experiment 1 at 24hpi is shown, however population distributions were generated at both time points, for all biological replicates.</p

Modulation of interleukin-1β-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts

Introduction: Innate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1β-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis. Methods: Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1β in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) was monitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by using multiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impact on IL-1β-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1β-induced pathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using various immunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptide followed by microscopy probing with streptavidin-Alexa Fluor. Results: This study demonstrated that IDR-1002 suppressed the production of IL-1β-induced MMP-3 and monocyte chemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing all chemokine responses. IDR-1002 altered the IL-1β-induced proteome primarily by altering the expression of members of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data also suggested that IDR-1002 was altering the transcription factor HNF-4α-mediated responses, known to be critical in metabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1β-induced NF-κB, JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002. Conclusions: This study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1β-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. The results of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidates for chronic inflammatory diseases such as inflammatory arthritis.Other UBCNon UBCReviewedFacult