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Comparative proteomic analyses demonstrate enhanced interferon and STAT-1 activation in reovirus T3D-infected HeLa cells.

Ezzati P, Komher K, Severini G, Coombs KM - Front Cell Infect Microbiol (2015)

Bottom Line: Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively.Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1.This study expands our understanding of reovirus-induced host responses.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba Winnipeg, MB, Canada.

ABSTRACT
As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC) analyses of reovirus, strain type 3 Dearing (T3D)-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation) to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D). Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1. This study expands our understanding of reovirus-induced host responses.

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Related in: MedlinePlus

Molecular pathways of regulated proteins. Proteins and their levels of regulation were imported into the Ingenuity Pathways Analysis (IPA®) tool and interacting pathways were constructed. (A) Ontological classifications of all measured proteins (Total) as well as those significantly up- and down-regulated by each of the viruses. (B–E) The top four IPA networks, identified at 95% confidence and each of which contained 12 or more “focus” molecules (molecules significantly up- or down-regulated), with pathway names indicated. Solid lines: direct known interactions; dashed lines: suspected or indirect interactions. Significantly regulated proteins identified in either the cytosolic or nuclear fractions were overlaid onto each network; red, significantly up-regulated proteins; pink, moderately up-regulated proteins; gray, proteins identified but not significantly regulated; light green, moderately down-regulated proteins; dark green, significantly down-regulated proteins; white, proteins known to be in network, but not identified in our study. Molecular classes are indicated in legend. Additional networks, also with 12 or more “focus” molecules, are depicted in Supplementary Figure S1.
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Figure 3: Molecular pathways of regulated proteins. Proteins and their levels of regulation were imported into the Ingenuity Pathways Analysis (IPA®) tool and interacting pathways were constructed. (A) Ontological classifications of all measured proteins (Total) as well as those significantly up- and down-regulated by each of the viruses. (B–E) The top four IPA networks, identified at 95% confidence and each of which contained 12 or more “focus” molecules (molecules significantly up- or down-regulated), with pathway names indicated. Solid lines: direct known interactions; dashed lines: suspected or indirect interactions. Significantly regulated proteins identified in either the cytosolic or nuclear fractions were overlaid onto each network; red, significantly up-regulated proteins; pink, moderately up-regulated proteins; gray, proteins identified but not significantly regulated; light green, moderately down-regulated proteins; dark green, significantly down-regulated proteins; white, proteins known to be in network, but not identified in our study. Molecular classes are indicated in legend. Additional networks, also with 12 or more “focus” molecules, are depicted in Supplementary Figure S1.

Mentions: The differentially-regulated proteins were analyzed by a variety of software tools. Ingenuity Pathway Analyses (IPA) globally mapped all genes into cytokines, enzymes, growth factors, and other categories (Figure 3A). There were significant differences in the proportions of enzymes, transcription regulators, transporters, and other GO classes differentially regulated by the three virus treatments (Figure 3A). Mapping up- and down-regulated proteins into IPA networks identified nine networks that contained at least 12 focus molecules. The four highest scoring networks were: cell signaling, dermatological diseases and conditions, antimicrobial response (Figure 3B); Organismal development, RNA post-transcriptional modification, cardiovascular disease (Figure 3C); Cell death and survival, cell signaling, small molecule biochemistry (Figure 3D); and Gene expression, cell cycle, infectious diseases (Figure 3E). Additional major networks included: Cancer, Immunological disease; Inflammatory disease and response; Tissue development; DNA replication; and Cell cycle and cellular development (Supplementary Figure S1). All networks showed significant differences in the specific members that were up-regulated, non-regulated, or down-regulated by T1L, T3D, or UV-T3D. Similarly, IPA analysis identified numerous significantly-affected canonical pathways. The Interferon signaling pathway was differentially affected by the three different virus treatments (Figure 4). As reflected by differences in respective protein levels (Table 1), numerous members of this pathway were up-regulated by T3D infection but not by T1L infection, and Mx1 and IFIT3 were down-regulated by UV-T3D treatment. Representative additional canonical pathways, such as Activation of IRF by cytosolic pattern recognition receptors, EIF2 signaling, ILK signaling, and Mitochondrial dysfunction, were also differentially regulated by the three different virus treatments (Supplementary Figure S2).


Comparative proteomic analyses demonstrate enhanced interferon and STAT-1 activation in reovirus T3D-infected HeLa cells.

Ezzati P, Komher K, Severini G, Coombs KM - Front Cell Infect Microbiol (2015)

Molecular pathways of regulated proteins. Proteins and their levels of regulation were imported into the Ingenuity Pathways Analysis (IPA®) tool and interacting pathways were constructed. (A) Ontological classifications of all measured proteins (Total) as well as those significantly up- and down-regulated by each of the viruses. (B–E) The top four IPA networks, identified at 95% confidence and each of which contained 12 or more “focus” molecules (molecules significantly up- or down-regulated), with pathway names indicated. Solid lines: direct known interactions; dashed lines: suspected or indirect interactions. Significantly regulated proteins identified in either the cytosolic or nuclear fractions were overlaid onto each network; red, significantly up-regulated proteins; pink, moderately up-regulated proteins; gray, proteins identified but not significantly regulated; light green, moderately down-regulated proteins; dark green, significantly down-regulated proteins; white, proteins known to be in network, but not identified in our study. Molecular classes are indicated in legend. Additional networks, also with 12 or more “focus” molecules, are depicted in Supplementary Figure S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388007&req=5

Figure 3: Molecular pathways of regulated proteins. Proteins and their levels of regulation were imported into the Ingenuity Pathways Analysis (IPA®) tool and interacting pathways were constructed. (A) Ontological classifications of all measured proteins (Total) as well as those significantly up- and down-regulated by each of the viruses. (B–E) The top four IPA networks, identified at 95% confidence and each of which contained 12 or more “focus” molecules (molecules significantly up- or down-regulated), with pathway names indicated. Solid lines: direct known interactions; dashed lines: suspected or indirect interactions. Significantly regulated proteins identified in either the cytosolic or nuclear fractions were overlaid onto each network; red, significantly up-regulated proteins; pink, moderately up-regulated proteins; gray, proteins identified but not significantly regulated; light green, moderately down-regulated proteins; dark green, significantly down-regulated proteins; white, proteins known to be in network, but not identified in our study. Molecular classes are indicated in legend. Additional networks, also with 12 or more “focus” molecules, are depicted in Supplementary Figure S1.
Mentions: The differentially-regulated proteins were analyzed by a variety of software tools. Ingenuity Pathway Analyses (IPA) globally mapped all genes into cytokines, enzymes, growth factors, and other categories (Figure 3A). There were significant differences in the proportions of enzymes, transcription regulators, transporters, and other GO classes differentially regulated by the three virus treatments (Figure 3A). Mapping up- and down-regulated proteins into IPA networks identified nine networks that contained at least 12 focus molecules. The four highest scoring networks were: cell signaling, dermatological diseases and conditions, antimicrobial response (Figure 3B); Organismal development, RNA post-transcriptional modification, cardiovascular disease (Figure 3C); Cell death and survival, cell signaling, small molecule biochemistry (Figure 3D); and Gene expression, cell cycle, infectious diseases (Figure 3E). Additional major networks included: Cancer, Immunological disease; Inflammatory disease and response; Tissue development; DNA replication; and Cell cycle and cellular development (Supplementary Figure S1). All networks showed significant differences in the specific members that were up-regulated, non-regulated, or down-regulated by T1L, T3D, or UV-T3D. Similarly, IPA analysis identified numerous significantly-affected canonical pathways. The Interferon signaling pathway was differentially affected by the three different virus treatments (Figure 4). As reflected by differences in respective protein levels (Table 1), numerous members of this pathway were up-regulated by T3D infection but not by T1L infection, and Mx1 and IFIT3 were down-regulated by UV-T3D treatment. Representative additional canonical pathways, such as Activation of IRF by cytosolic pattern recognition receptors, EIF2 signaling, ILK signaling, and Mitochondrial dysfunction, were also differentially regulated by the three different virus treatments (Supplementary Figure S2).

Bottom Line: Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively.Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1.This study expands our understanding of reovirus-induced host responses.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba Winnipeg, MB, Canada.

ABSTRACT
As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC) analyses of reovirus, strain type 3 Dearing (T3D)-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation) to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D). Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1. This study expands our understanding of reovirus-induced host responses.

Show MeSH
Related in: MedlinePlus