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Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase.

Tange S, Zhou Y, Nagakui-Noguchi Y, Imai T, Nakanishi A - Virol. J. (2013)

Bottom Line: Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production.Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1.Our results also reveal the role of PKA in viral production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Gene Therapy, Department of Aging Intervention, National Center for Geriatrics and Gerontology, 35, Gengo, Morioka, Obu, Aichi 474-8522, Japan.

ABSTRACT

Background: Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear.

Methods: A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells.

Results: Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production.

Conclusions: Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.

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Related in: MedlinePlus

Effects of kinase inhibitors on capsid protein expression following HAstV1 infection. (A) Caco-2 cells were infected with HAstV1 (MOI = 0.22) in the presence of solvent alone (DMSO, panels A and a), staurosporine (4 μM; B and b), genistein (10 μM; C and c), U0126 (20 μM; D and d), SB 203580 (50 μM; E and e), or JNK inhibitor II (50 μM; F and f). The cells were fixed at 24 h post-infection (hpi), and then HAstV capsid protein was detected by immunofluorescence. Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (anti-HAstV, A through F) and for cellular DNA (DAPI, a through f). (B) Caco-2 cells were infected with HAstV1 in the presence of various kinase inhibitors, LY294002 (50 μM; panels A and a), wortmannin (1 μM; B and b), Triciribine, (10 μM; C and c), MK-2206 (10 μM; D and d), NSC23766 (50 μM; E and e), Y27632 (50 μM; F and f), and H89 (10 μM; G and g). Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (A through G) and for cellular DNA (a through g). (C) Viability of Caco-2 cells infected with HAstV1 in the presence of designated drugs was examined by colorimetric assay using WST-1 reagent (Takara). The absorbance of the cell culture medium was measured at 450 nm versus a 650-nm reference. The vertical axis indicates arbitrary unit divided by the mean value of a solvent-only (DMSO) control sample. The mean value obtained using three of each sample is presented as bar graph, with the standard deviation indicated as error bar. Statistical significance, compared with the solvent control (DMSO) is indicated (*P < 0.05; **P < 0.01).
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Figure 1: Effects of kinase inhibitors on capsid protein expression following HAstV1 infection. (A) Caco-2 cells were infected with HAstV1 (MOI = 0.22) in the presence of solvent alone (DMSO, panels A and a), staurosporine (4 μM; B and b), genistein (10 μM; C and c), U0126 (20 μM; D and d), SB 203580 (50 μM; E and e), or JNK inhibitor II (50 μM; F and f). The cells were fixed at 24 h post-infection (hpi), and then HAstV capsid protein was detected by immunofluorescence. Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (anti-HAstV, A through F) and for cellular DNA (DAPI, a through f). (B) Caco-2 cells were infected with HAstV1 in the presence of various kinase inhibitors, LY294002 (50 μM; panels A and a), wortmannin (1 μM; B and b), Triciribine, (10 μM; C and c), MK-2206 (10 μM; D and d), NSC23766 (50 μM; E and e), Y27632 (50 μM; F and f), and H89 (10 μM; G and g). Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (A through G) and for cellular DNA (a through g). (C) Viability of Caco-2 cells infected with HAstV1 in the presence of designated drugs was examined by colorimetric assay using WST-1 reagent (Takara). The absorbance of the cell culture medium was measured at 450 nm versus a 650-nm reference. The vertical axis indicates arbitrary unit divided by the mean value of a solvent-only (DMSO) control sample. The mean value obtained using three of each sample is presented as bar graph, with the standard deviation indicated as error bar. Statistical significance, compared with the solvent control (DMSO) is indicated (*P < 0.05; **P < 0.01).

Mentions: To search for the signaling pathways that are important for HAstV1 infection, we examined various kinase blockers inhibitors (Table 1) for their ability to block HAstV1 infection of Caco-2 cells. Caco-2 cells were infected with HAstV1 in the presence or absence of each kinase inhibitor, and the presence of the inhibitor was maintained until 24 hours post-infection (hpi), when the cells were detected for viral capsid protein by immunofluorescence. While DMSO, the solvent for the inhibitors, did not interfere with viral gene expression (Figure 1A, panels A and a), 4 μM staurosporine (Figure 1A, B and b), a general kinase inhibitor, or 10 μM genistein (Figure 1A, C and c), a general inhibitor for tyrosine kinases, blocked viral gene expression. We noted that staurosporine treatment caused modest cellular toxicity, evident by nuclear staining with DAPI (Figure 1A, b) and by colorimetric assay for cell viability (Figure 1C). However, the almost complete absence of cells positive for viral antigen suggests that the drug was effective in blocking infection in the cells that survived drug treatment. Consistent with the previously reported requirement for ERK1/2 signaling in HAstV1 infection [19], U0126, a MEK1/2 inhibitor that blocks ERK1/2 phosphorylation, also blocked viral gene expression (Figure 1A, D and d). Other members of the MAPK family that we tested did not appear to be involved in establishing HAstV1 infection because neither 50 μM SB 203580, which blocks p38 activation (Figure 1A, E and e), nor 50 μM JNK inhibitor II, which selectively inhibits JNK (Figure 1A, F and f), had a significant effect on viral capsid gene expression.


Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase.

Tange S, Zhou Y, Nagakui-Noguchi Y, Imai T, Nakanishi A - Virol. J. (2013)

Effects of kinase inhibitors on capsid protein expression following HAstV1 infection. (A) Caco-2 cells were infected with HAstV1 (MOI = 0.22) in the presence of solvent alone (DMSO, panels A and a), staurosporine (4 μM; B and b), genistein (10 μM; C and c), U0126 (20 μM; D and d), SB 203580 (50 μM; E and e), or JNK inhibitor II (50 μM; F and f). The cells were fixed at 24 h post-infection (hpi), and then HAstV capsid protein was detected by immunofluorescence. Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (anti-HAstV, A through F) and for cellular DNA (DAPI, a through f). (B) Caco-2 cells were infected with HAstV1 in the presence of various kinase inhibitors, LY294002 (50 μM; panels A and a), wortmannin (1 μM; B and b), Triciribine, (10 μM; C and c), MK-2206 (10 μM; D and d), NSC23766 (50 μM; E and e), Y27632 (50 μM; F and f), and H89 (10 μM; G and g). Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (A through G) and for cellular DNA (a through g). (C) Viability of Caco-2 cells infected with HAstV1 in the presence of designated drugs was examined by colorimetric assay using WST-1 reagent (Takara). The absorbance of the cell culture medium was measured at 450 nm versus a 650-nm reference. The vertical axis indicates arbitrary unit divided by the mean value of a solvent-only (DMSO) control sample. The mean value obtained using three of each sample is presented as bar graph, with the standard deviation indicated as error bar. Statistical significance, compared with the solvent control (DMSO) is indicated (*P < 0.05; **P < 0.01).
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Related In: Results  -  Collection

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Figure 1: Effects of kinase inhibitors on capsid protein expression following HAstV1 infection. (A) Caco-2 cells were infected with HAstV1 (MOI = 0.22) in the presence of solvent alone (DMSO, panels A and a), staurosporine (4 μM; B and b), genistein (10 μM; C and c), U0126 (20 μM; D and d), SB 203580 (50 μM; E and e), or JNK inhibitor II (50 μM; F and f). The cells were fixed at 24 h post-infection (hpi), and then HAstV capsid protein was detected by immunofluorescence. Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (anti-HAstV, A through F) and for cellular DNA (DAPI, a through f). (B) Caco-2 cells were infected with HAstV1 in the presence of various kinase inhibitors, LY294002 (50 μM; panels A and a), wortmannin (1 μM; B and b), Triciribine, (10 μM; C and c), MK-2206 (10 μM; D and d), NSC23766 (50 μM; E and e), Y27632 (50 μM; F and f), and H89 (10 μM; G and g). Each pair of panels shows, for the same field of cells, the staining patterns for the viral capsid protein (A through G) and for cellular DNA (a through g). (C) Viability of Caco-2 cells infected with HAstV1 in the presence of designated drugs was examined by colorimetric assay using WST-1 reagent (Takara). The absorbance of the cell culture medium was measured at 450 nm versus a 650-nm reference. The vertical axis indicates arbitrary unit divided by the mean value of a solvent-only (DMSO) control sample. The mean value obtained using three of each sample is presented as bar graph, with the standard deviation indicated as error bar. Statistical significance, compared with the solvent control (DMSO) is indicated (*P < 0.05; **P < 0.01).
Mentions: To search for the signaling pathways that are important for HAstV1 infection, we examined various kinase blockers inhibitors (Table 1) for their ability to block HAstV1 infection of Caco-2 cells. Caco-2 cells were infected with HAstV1 in the presence or absence of each kinase inhibitor, and the presence of the inhibitor was maintained until 24 hours post-infection (hpi), when the cells were detected for viral capsid protein by immunofluorescence. While DMSO, the solvent for the inhibitors, did not interfere with viral gene expression (Figure 1A, panels A and a), 4 μM staurosporine (Figure 1A, B and b), a general kinase inhibitor, or 10 μM genistein (Figure 1A, C and c), a general inhibitor for tyrosine kinases, blocked viral gene expression. We noted that staurosporine treatment caused modest cellular toxicity, evident by nuclear staining with DAPI (Figure 1A, b) and by colorimetric assay for cell viability (Figure 1C). However, the almost complete absence of cells positive for viral antigen suggests that the drug was effective in blocking infection in the cells that survived drug treatment. Consistent with the previously reported requirement for ERK1/2 signaling in HAstV1 infection [19], U0126, a MEK1/2 inhibitor that blocks ERK1/2 phosphorylation, also blocked viral gene expression (Figure 1A, D and d). Other members of the MAPK family that we tested did not appear to be involved in establishing HAstV1 infection because neither 50 μM SB 203580, which blocks p38 activation (Figure 1A, E and e), nor 50 μM JNK inhibitor II, which selectively inhibits JNK (Figure 1A, F and f), had a significant effect on viral capsid gene expression.

Bottom Line: Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production.Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1.Our results also reveal the role of PKA in viral production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Gene Therapy, Department of Aging Intervention, National Center for Geriatrics and Gerontology, 35, Gengo, Morioka, Obu, Aichi 474-8522, Japan.

ABSTRACT

Background: Upon initial contact with a virus, host cells activate a series of cellular signaling cascades that facilitate viral entry and viral propagation within the cell. Little is known about how the human astrovirus (HAstV) exploits signaling cascades to establish an infection in host cells. Recent studies showed that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is important for HAstV infection, though the involvement of other signaling cascades remains unclear.

Methods: A panel of kinase blockers was used to search for cellular signaling pathways important for HAstV1 infection. To determine their impact on the infectious process, we examined viral gene expression, RNA replication, and viral RNA and capsid protein release from host cells.

Results: Inhibitors of phosphoinositide 3-kinase (PI3K) activation interfered with the infection, independent of their effect on ERK 1/2 activation. Activation of the PI3K signaling cascade occurred at an early phase of the infection, judging from the timeframe of Akt phosphorylation. PI3K inhibition at early times, but not at later times, blocked viral gene expression. However, inhibiting the downstream targets of PI3K activation, Akt and Rac1, did not block infection. Inhibition of protein kinase A (PKA) activation was found to block a later phase of HAstV1 production.

Conclusions: Our results reveal a previously unknown, essential role of PI3K in the life cycle of HAstV1. PI3K participates in the early stage of infection, possibly during the viral entry process. Our results also reveal the role of PKA in viral production.

Show MeSH
Related in: MedlinePlus