Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunoblot analyses of indicated host proteins in HeLa cells mock-infected, or infected with T3D or T1L, or treated with UV-inactivated T3D. Cells were harvested and lysed with 0.5% NP-40 detergent, nuclei removed, and cytosolic fractions dissolved in SDS electrophoresis sample buffer, resolved in 10% mini SDS-PAGE, transferred to PVDF, and probed with various antibodies. Bands were visualized, and intensities measured, with an Alpha Innotech FluorChem®Q MultiImage® III instrument.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4388007&req=5

Figure 2: Immunoblot analyses of indicated host proteins in HeLa cells mock-infected, or infected with T3D or T1L, or treated with UV-inactivated T3D. Cells were harvested and lysed with 0.5% NP-40 detergent, nuclei removed, and cytosolic fractions dissolved in SDS electrophoresis sample buffer, resolved in 10% mini SDS-PAGE, transferred to PVDF, and probed with various antibodies. Bands were visualized, and intensities measured, with an Alpha Innotech FluorChem®Q MultiImage® III instrument.

Mentions: To confirm some of the iTRAQ-determined protein ratios, we analyzed selected proteins in T1L- and T3D-infected, and UV-T3D-treated, cells. Immunoblotting confirmed that STAT-1, ISG15, IFIT1, and Mx1 were strongly up-regulated in T3D-infected cells but only weakly up-regulated in corresponding T1L-infected and UV-T3D-treated cells (Figure 2).


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)

Immunoblot analyses of indicated host proteins in HeLa cells mock-infected, or infected with T3D or T1L, or treated with UV-inactivated T3D. Cells were harvested and lysed with 0.5% NP-40 detergent, nuclei removed, and cytosolic fractions dissolved in SDS electrophoresis sample buffer, resolved in 10% mini SDS-PAGE, transferred to PVDF, and probed with various antibodies. Bands were visualized, and intensities measured, with an Alpha Innotech FluorChem®Q MultiImage® III instrument.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Immunoblot analyses of indicated host proteins in HeLa cells mock-infected, or infected with T3D or T1L, or treated with UV-inactivated T3D. Cells were harvested and lysed with 0.5% NP-40 detergent, nuclei removed, and cytosolic fractions dissolved in SDS electrophoresis sample buffer, resolved in 10% mini SDS-PAGE, transferred to PVDF, and probed with various antibodies. Bands were visualized, and intensities measured, with an Alpha Innotech FluorChem®Q MultiImage® III instrument.
Mentions: To confirm some of the iTRAQ-determined protein ratios, we analyzed selected proteins in T1L- and T3D-infected, and UV-T3D-treated, cells. Immunoblotting confirmed that STAT-1, ISG15, IFIT1, and Mx1 were strongly up-regulated in T3D-infected cells but only weakly up-regulated in corresponding T1L-infected and UV-T3D-treated cells (Figure 2).

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