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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

Distributions of proteins identified in various experiments. (A) Venn diagrams of numbers of identified proteins from various experimental replicates and cell fractions. Cells that were mock infected, or infected with reovirus T1L or T3D, or treated with UV-inactivated T3D, were harvested at 24 hpi, fractionated into the cytosolic (Cyto, left) and nuclei (Nuc, center) fractions and labeled with iTRAQ reagents. Numbers of proteins identified with two or more peptides and at >99% confidence are indicated for each of the three experimental replicates. The total numbers of unique proteins identified in the cytosolic and nuclear fractions are indicated at right. (B) Frequency distributions of identified proteins and their expression ratios (expressed as Log2 fold change compared to Mock) in representative 8-plex iTRAQ samples. The two mock samples were compared to each other and to each of the other six treatments. Standard deviations of the variance in each cytoplasmic (left) and nuclear (right) sample are shown in the box in each plot. The two thin vertical lines represent ±7 σ, corresponding to the limits within which virtually all mock samples fall. Positive values represent up-regulated host proteins in virus-infected cells; negative values represent down-regulated host proteins. Only the distributions of one set of each treatment are shown for clarity.
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Figure 1: Distributions of proteins identified in various experiments. (A) Venn diagrams of numbers of identified proteins from various experimental replicates and cell fractions. Cells that were mock infected, or infected with reovirus T1L or T3D, or treated with UV-inactivated T3D, were harvested at 24 hpi, fractionated into the cytosolic (Cyto, left) and nuclei (Nuc, center) fractions and labeled with iTRAQ reagents. Numbers of proteins identified with two or more peptides and at >99% confidence are indicated for each of the three experimental replicates. The total numbers of unique proteins identified in the cytosolic and nuclear fractions are indicated at right. (B) Frequency distributions of identified proteins and their expression ratios (expressed as Log2 fold change compared to Mock) in representative 8-plex iTRAQ samples. The two mock samples were compared to each other and to each of the other six treatments. Standard deviations of the variance in each cytoplasmic (left) and nuclear (right) sample are shown in the box in each plot. The two thin vertical lines represent ±7 σ, corresponding to the limits within which virtually all mock samples fall. Positive values represent up-regulated host proteins in virus-infected cells; negative values represent down-regulated host proteins. Only the distributions of one set of each treatment are shown for clarity.

Mentions: Titration of the gradient-purified virions and comparisons to optically-determined particle counts indicated that the particle-to-PFU ratios of the various virus samples were: T1L: 191; T3D: 327; and UV-treated T3D: >3.5 × 108, respectively. These different virus preparations were then used to infect HeLa cells, cells were harvested at 24 hpi, and cytosolic and nuclear fractions prepared and analyzed as described in Materials and Methods. After removal of proteins identified at <99% confidence and those identified by a single peptide, a total of 1562, 1794, and 1599 proteins were detected and relative quantities measured in the two 4-plex and one 8-plex cytosolic fractions, respectively, and 654, 650, and 782 proteins were measured in the corresponding nuclear fractions (Figure 1A, left and center). 1491 proteins were found exclusively in the cytosol, 340 were found just in the nucleus, and 544 were found in both fractions (Figure 1A, right). Experimental variability was also assessed by examination of the 8-plex technical replicates. The two mock samples were compared to each other and to the various infections (Figure 1B). The standard deviations of Infected:Mock Log2 ratios were 0.051 and 0.034 for the cytosolic and nuclear Mock replicates, respectively, and 3.9- to 14-fold higher for each of the infected samples, indicating that host protein ratios were greatly affected by the virus treatments.


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)

Distributions of proteins identified in various experiments. (A) Venn diagrams of numbers of identified proteins from various experimental replicates and cell fractions. Cells that were mock infected, or infected with reovirus T1L or T3D, or treated with UV-inactivated T3D, were harvested at 24 hpi, fractionated into the cytosolic (Cyto, left) and nuclei (Nuc, center) fractions and labeled with iTRAQ reagents. Numbers of proteins identified with two or more peptides and at >99% confidence are indicated for each of the three experimental replicates. The total numbers of unique proteins identified in the cytosolic and nuclear fractions are indicated at right. (B) Frequency distributions of identified proteins and their expression ratios (expressed as Log2 fold change compared to Mock) in representative 8-plex iTRAQ samples. The two mock samples were compared to each other and to each of the other six treatments. Standard deviations of the variance in each cytoplasmic (left) and nuclear (right) sample are shown in the box in each plot. The two thin vertical lines represent ±7 σ, corresponding to the limits within which virtually all mock samples fall. Positive values represent up-regulated host proteins in virus-infected cells; negative values represent down-regulated host proteins. Only the distributions of one set of each treatment are shown for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4388007&req=5

Figure 1: Distributions of proteins identified in various experiments. (A) Venn diagrams of numbers of identified proteins from various experimental replicates and cell fractions. Cells that were mock infected, or infected with reovirus T1L or T3D, or treated with UV-inactivated T3D, were harvested at 24 hpi, fractionated into the cytosolic (Cyto, left) and nuclei (Nuc, center) fractions and labeled with iTRAQ reagents. Numbers of proteins identified with two or more peptides and at >99% confidence are indicated for each of the three experimental replicates. The total numbers of unique proteins identified in the cytosolic and nuclear fractions are indicated at right. (B) Frequency distributions of identified proteins and their expression ratios (expressed as Log2 fold change compared to Mock) in representative 8-plex iTRAQ samples. The two mock samples were compared to each other and to each of the other six treatments. Standard deviations of the variance in each cytoplasmic (left) and nuclear (right) sample are shown in the box in each plot. The two thin vertical lines represent ±7 σ, corresponding to the limits within which virtually all mock samples fall. Positive values represent up-regulated host proteins in virus-infected cells; negative values represent down-regulated host proteins. Only the distributions of one set of each treatment are shown for clarity.
Mentions: Titration of the gradient-purified virions and comparisons to optically-determined particle counts indicated that the particle-to-PFU ratios of the various virus samples were: T1L: 191; T3D: 327; and UV-treated T3D: >3.5 × 108, respectively. These different virus preparations were then used to infect HeLa cells, cells were harvested at 24 hpi, and cytosolic and nuclear fractions prepared and analyzed as described in Materials and Methods. After removal of proteins identified at <99% confidence and those identified by a single peptide, a total of 1562, 1794, and 1599 proteins were detected and relative quantities measured in the two 4-plex and one 8-plex cytosolic fractions, respectively, and 654, 650, and 782 proteins were measured in the corresponding nuclear fractions (Figure 1A, left and center). 1491 proteins were found exclusively in the cytosol, 340 were found just in the nucleus, and 544 were found in both fractions (Figure 1A, right). Experimental variability was also assessed by examination of the 8-plex technical replicates. The two mock samples were compared to each other and to the various infections (Figure 1B). The standard deviations of Infected:Mock Log2 ratios were 0.051 and 0.034 for the cytosolic and nuclear Mock replicates, respectively, and 3.9- to 14-fold higher for each of the infected samples, indicating that host protein ratios were greatly affected by the virus treatments.

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