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Proteolytic disassembly of viral outer capsid proteins is crucial for reovirus-mediated type-I interferon induction in both reovirus-susceptible and reovirus-refractory tumor cells.

Katayama Y, Terasawa Y, Tachibana M, Mizuguchi H, Sakurai F - Biomed Res Int (2015)

Bottom Line: Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application.In this study, we examined reovirus-induced upregulation of interferon- (IFN-) β and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells.These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application. Reovirus-induced innate immune responses have been relatively well characterized in immune cells and mouse embryonic fibroblasts cells; however, the mechanisms and profiles of reovirus-induced innate immune responses in human tumor cells have not been well understood. In particular, differences in reovirus-induced innate immune responses between reovirus-susceptible and reovirus-refractory tumor cells remain unknown, although the intracellular trafficking of reovirus differs between these tumor cells. In this study, we examined reovirus-induced upregulation of interferon- (IFN-) β and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells. IFN-β and Noxa were significantly induced by reovirus via the IFN-β promoter stimulator-1 (IPS-1) signaling in both types of tumor cells. Inhibition of cathepsins B and L, which are important for disassembly of reovirus outer capsid proteins and escape into cytoplasm, largely suppressed reovirus-induced upregulation of IFN-β and Noxa expression in not only reovirus-susceptible but also reovirus-refractory tumor cells. These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.

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Effects of knockdown of RNA sensors and their adaptor molecules on reovirus-induced expression of IFN-β and Noxa in tumor cells. (a) Knockdown efficiencies of siRNAs. Cells were transfected with siRNAs targeting indicated gene or control siRNA (siControl). Following a 48-hour incubation, cells were harvested for the evaluation of siRNA-mediated knockdown efficiencies by qRT-PCR analysis. (b) mRNA levels of IFN-β and Noxa following reovirus infection in the cells pretreated with siRNAs. Forty-eight hours after transfection with siRNAs, cells were infected with reovirus at an MOI of 20. Cells were harvested 24 hrs after infection for analysis of IFN-β and Noxa mRNA levels by qRT-PCR analysis. The data were normalized by the data of the siControl group infected with reovirus. The data shown represent the mean ± SD of triplicate measurements. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the siControl group infected with reovirus.
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fig2: Effects of knockdown of RNA sensors and their adaptor molecules on reovirus-induced expression of IFN-β and Noxa in tumor cells. (a) Knockdown efficiencies of siRNAs. Cells were transfected with siRNAs targeting indicated gene or control siRNA (siControl). Following a 48-hour incubation, cells were harvested for the evaluation of siRNA-mediated knockdown efficiencies by qRT-PCR analysis. (b) mRNA levels of IFN-β and Noxa following reovirus infection in the cells pretreated with siRNAs. Forty-eight hours after transfection with siRNAs, cells were infected with reovirus at an MOI of 20. Cells were harvested 24 hrs after infection for analysis of IFN-β and Noxa mRNA levels by qRT-PCR analysis. The data were normalized by the data of the siControl group infected with reovirus. The data shown represent the mean ± SD of triplicate measurements. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the siControl group infected with reovirus.

Mentions: Next, in order to examine which RNA sensors are involved in reovirus-mediated induction of IFN-β and Noxa in tumor cells, tumor cells were transfected with siRNAs against various types of RNA sensors, followed by infection with reovirus. Although several studies demonstrated that the reovirus genome is recognized by cytoplasmic dsRNA sensors RIG-I and MDA5 in immune cells and mouse embryonic fibroblasts [15], DExD/H box helicase families, which are involved in the recognition of double-stranded RNA, were recently identified [16–19]. Expression of the RNA sensor genes was knocked down significantly, by more than 50%, following siRNA transfection (Figure 2(a)). We confirmed that treatment with siRNA alone did not induce upregulation of IFN-β mRNA levels (data not shown). Compared with the control siRNA group, reovirus-induced IFN-β mRNA levels were clearly decreased in the cells transfected with siRNAs against RIG-I (siRIG-I) and IPS-1 (siIPS-1) in H1299 and A549 cells (Figure 2(b)). In HepG2 cells, knockdown of not only RIG-I and IPS-1 but also MyD88, DDX1, DHX9, and PKR resulted in a significant decrease of IFN-β mRNA levels. In A431 cells, reovirus-induced IFN-β mRNA levels were decreased by knockdown of IPS-1 and PKR but not RIG-I. In all the cells examined, reovirus-induced upregulation of IFN-β expression was most largely suppressed by knockdown of IPS-1 among the molecules examined in this study. In contrast, IFN-β mRNA levels were comparable or higher in the cells transfected with siRNAs against MDA5 and several of the RNA sensors and adaptor molecules compared to those in the cells transfected with control siRNAs. Noxa mRNA profiles were similar to those of IFN-β mRNA in H1299, HepG2, and A549 cells, although pretreatment with siRNAs against the DExD/H box helicase families did not result in significant increases in reovirus-induced Noxa mRNA levels in H1299 and A549 cells. Knockdown of IPS-1 induced the largest reduction in the Noxa mRNA levels in all three cell lines. These results indicated that, regardless of susceptibility to reovirus, the IPS-1-dependent pathway was mainly involved in reovirus-mediated induction of IFN-β and Noxa in tumor cells. Effects of knockdown of RNA sensors and adaptor molecules on reovirus-mediated Noxa induction were not examined in A431 cells, because infection with reovirus at an MOI of 20 did not result in a statistically significant elevation in the Noxa mRNA levels in A431 cells, as shown in Figure 1.


Proteolytic disassembly of viral outer capsid proteins is crucial for reovirus-mediated type-I interferon induction in both reovirus-susceptible and reovirus-refractory tumor cells.

Katayama Y, Terasawa Y, Tachibana M, Mizuguchi H, Sakurai F - Biomed Res Int (2015)

Effects of knockdown of RNA sensors and their adaptor molecules on reovirus-induced expression of IFN-β and Noxa in tumor cells. (a) Knockdown efficiencies of siRNAs. Cells were transfected with siRNAs targeting indicated gene or control siRNA (siControl). Following a 48-hour incubation, cells were harvested for the evaluation of siRNA-mediated knockdown efficiencies by qRT-PCR analysis. (b) mRNA levels of IFN-β and Noxa following reovirus infection in the cells pretreated with siRNAs. Forty-eight hours after transfection with siRNAs, cells were infected with reovirus at an MOI of 20. Cells were harvested 24 hrs after infection for analysis of IFN-β and Noxa mRNA levels by qRT-PCR analysis. The data were normalized by the data of the siControl group infected with reovirus. The data shown represent the mean ± SD of triplicate measurements. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the siControl group infected with reovirus.
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Related In: Results  -  Collection

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fig2: Effects of knockdown of RNA sensors and their adaptor molecules on reovirus-induced expression of IFN-β and Noxa in tumor cells. (a) Knockdown efficiencies of siRNAs. Cells were transfected with siRNAs targeting indicated gene or control siRNA (siControl). Following a 48-hour incubation, cells were harvested for the evaluation of siRNA-mediated knockdown efficiencies by qRT-PCR analysis. (b) mRNA levels of IFN-β and Noxa following reovirus infection in the cells pretreated with siRNAs. Forty-eight hours after transfection with siRNAs, cells were infected with reovirus at an MOI of 20. Cells were harvested 24 hrs after infection for analysis of IFN-β and Noxa mRNA levels by qRT-PCR analysis. The data were normalized by the data of the siControl group infected with reovirus. The data shown represent the mean ± SD of triplicate measurements. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the siControl group infected with reovirus.
Mentions: Next, in order to examine which RNA sensors are involved in reovirus-mediated induction of IFN-β and Noxa in tumor cells, tumor cells were transfected with siRNAs against various types of RNA sensors, followed by infection with reovirus. Although several studies demonstrated that the reovirus genome is recognized by cytoplasmic dsRNA sensors RIG-I and MDA5 in immune cells and mouse embryonic fibroblasts [15], DExD/H box helicase families, which are involved in the recognition of double-stranded RNA, were recently identified [16–19]. Expression of the RNA sensor genes was knocked down significantly, by more than 50%, following siRNA transfection (Figure 2(a)). We confirmed that treatment with siRNA alone did not induce upregulation of IFN-β mRNA levels (data not shown). Compared with the control siRNA group, reovirus-induced IFN-β mRNA levels were clearly decreased in the cells transfected with siRNAs against RIG-I (siRIG-I) and IPS-1 (siIPS-1) in H1299 and A549 cells (Figure 2(b)). In HepG2 cells, knockdown of not only RIG-I and IPS-1 but also MyD88, DDX1, DHX9, and PKR resulted in a significant decrease of IFN-β mRNA levels. In A431 cells, reovirus-induced IFN-β mRNA levels were decreased by knockdown of IPS-1 and PKR but not RIG-I. In all the cells examined, reovirus-induced upregulation of IFN-β expression was most largely suppressed by knockdown of IPS-1 among the molecules examined in this study. In contrast, IFN-β mRNA levels were comparable or higher in the cells transfected with siRNAs against MDA5 and several of the RNA sensors and adaptor molecules compared to those in the cells transfected with control siRNAs. Noxa mRNA profiles were similar to those of IFN-β mRNA in H1299, HepG2, and A549 cells, although pretreatment with siRNAs against the DExD/H box helicase families did not result in significant increases in reovirus-induced Noxa mRNA levels in H1299 and A549 cells. Knockdown of IPS-1 induced the largest reduction in the Noxa mRNA levels in all three cell lines. These results indicated that, regardless of susceptibility to reovirus, the IPS-1-dependent pathway was mainly involved in reovirus-mediated induction of IFN-β and Noxa in tumor cells. Effects of knockdown of RNA sensors and adaptor molecules on reovirus-mediated Noxa induction were not examined in A431 cells, because infection with reovirus at an MOI of 20 did not result in a statistically significant elevation in the Noxa mRNA levels in A431 cells, as shown in Figure 1.

Bottom Line: Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application.In this study, we examined reovirus-induced upregulation of interferon- (IFN-) β and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells.These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application. Reovirus-induced innate immune responses have been relatively well characterized in immune cells and mouse embryonic fibroblasts cells; however, the mechanisms and profiles of reovirus-induced innate immune responses in human tumor cells have not been well understood. In particular, differences in reovirus-induced innate immune responses between reovirus-susceptible and reovirus-refractory tumor cells remain unknown, although the intracellular trafficking of reovirus differs between these tumor cells. In this study, we examined reovirus-induced upregulation of interferon- (IFN-) β and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells. IFN-β and Noxa were significantly induced by reovirus via the IFN-β promoter stimulator-1 (IPS-1) signaling in both types of tumor cells. Inhibition of cathepsins B and L, which are important for disassembly of reovirus outer capsid proteins and escape into cytoplasm, largely suppressed reovirus-induced upregulation of IFN-β and Noxa expression in not only reovirus-susceptible but also reovirus-refractory tumor cells. These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity.

Show MeSH
Related in: MedlinePlus