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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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Effects of kinase blockers on viral RNA replication and on the appearance of viral RNA and capsid protein in culture supernatant. Caco-2 cells were infected with HAstV1 in the absence (Mock) or presence of various kinase inhibitors and examined for the following. (A)Effects on viral RNA replication. Viral RNA replication in the infected cells was analyzed using quantitative real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA, normalized to the amount of total RNA, was converted to a value relative to that of mock treatment. The mean relative value from four independent experiments, with the standard deviation indicated as error bar, is presented. (B)Effects on presence of viral RNA in culture supernatant. The level of viral RNA in the post-infection culture supernatant was analyzed using real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA was converted to a value relative to that of mock treatment. The mean relative value from three experiments, with the standard deviation indicated as error bar, is presented. (C)Effects on presence of viral capsid protein in culture supernatant. The level of viral capsid antigen in the post-infection culture supernatant was measured using ELISA. The supernatant samples were prepared in triplicate, and the mean capsid antigen level for each drug treatment was further converted to a value relative to mock treatment. Statistically significant differences, determined as in Figure 2D, are indicated on the graphs.
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Figure 3: Effects of kinase blockers on viral RNA replication and on the appearance of viral RNA and capsid protein in culture supernatant. Caco-2 cells were infected with HAstV1 in the absence (Mock) or presence of various kinase inhibitors and examined for the following. (A)Effects on viral RNA replication. Viral RNA replication in the infected cells was analyzed using quantitative real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA, normalized to the amount of total RNA, was converted to a value relative to that of mock treatment. The mean relative value from four independent experiments, with the standard deviation indicated as error bar, is presented. (B)Effects on presence of viral RNA in culture supernatant. The level of viral RNA in the post-infection culture supernatant was analyzed using real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA was converted to a value relative to that of mock treatment. The mean relative value from three experiments, with the standard deviation indicated as error bar, is presented. (C)Effects on presence of viral capsid protein in culture supernatant. The level of viral capsid antigen in the post-infection culture supernatant was measured using ELISA. The supernatant samples were prepared in triplicate, and the mean capsid antigen level for each drug treatment was further converted to a value relative to mock treatment. Statistically significant differences, determined as in Figure 2D, are indicated on the graphs.

Mentions: The immunofluorescence detection of viral capsid protein offered a qualitative indication of whether a given kinase inhibitor affected the initiation of the infection processes leading to viral gene expression. In order to more quantitatively measure the effect of the drugs on viral propagation, the amount of viral RNA produced in the cells at 24 hpi in the presence or absence of the drugs was measured by quantitative real-time RT-PCR (Figure 3A). Cells treated with genistein, staurosporine, U0126, and LY294002 contained significantly lower amounts of viral RNA than cells treated with the solvent alone, consistent with the finding that these drugs were inhibitory to the expression of viral capsid. Although treatment with wortmannin could show inhibitory effect on viral capsid expression (Figure 1B), it did not translate into a significant effect on viral RNA replication (see Discussion). Not surprisingly, drugs that did not inhibit viral gene expression—inhibitors of MAPK p38s (SB203580), JNK (JNK inhibitor II), Akt (MK2206), and PKA (H89)—had no measurable effect on the extent of viral RNA replication. Treatment with triciribine, NSC23766, or Y27632 induced higher levels of RNA replication and did not inhibit the production of viral RNA. These results support the idea that PI3K activation is important for the initiation of viral infection via a non-Akt, non-Rac mediated pathway.


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 blockers on viral RNA replication and on the appearance of viral RNA and capsid protein in culture supernatant. Caco-2 cells were infected with HAstV1 in the absence (Mock) or presence of various kinase inhibitors and examined for the following. (A)Effects on viral RNA replication. Viral RNA replication in the infected cells was analyzed using quantitative real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA, normalized to the amount of total RNA, was converted to a value relative to that of mock treatment. The mean relative value from four independent experiments, with the standard deviation indicated as error bar, is presented. (B)Effects on presence of viral RNA in culture supernatant. The level of viral RNA in the post-infection culture supernatant was analyzed using real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA was converted to a value relative to that of mock treatment. The mean relative value from three experiments, with the standard deviation indicated as error bar, is presented. (C)Effects on presence of viral capsid protein in culture supernatant. The level of viral capsid antigen in the post-infection culture supernatant was measured using ELISA. The supernatant samples were prepared in triplicate, and the mean capsid antigen level for each drug treatment was further converted to a value relative to mock treatment. Statistically significant differences, determined as in Figure 2D, are indicated on the graphs.
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Related In: Results  -  Collection

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Figure 3: Effects of kinase blockers on viral RNA replication and on the appearance of viral RNA and capsid protein in culture supernatant. Caco-2 cells were infected with HAstV1 in the absence (Mock) or presence of various kinase inhibitors and examined for the following. (A)Effects on viral RNA replication. Viral RNA replication in the infected cells was analyzed using quantitative real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA, normalized to the amount of total RNA, was converted to a value relative to that of mock treatment. The mean relative value from four independent experiments, with the standard deviation indicated as error bar, is presented. (B)Effects on presence of viral RNA in culture supernatant. The level of viral RNA in the post-infection culture supernatant was analyzed using real-time RT-PCR. For each drug treatment, the quantified amount of viral RNA was converted to a value relative to that of mock treatment. The mean relative value from three experiments, with the standard deviation indicated as error bar, is presented. (C)Effects on presence of viral capsid protein in culture supernatant. The level of viral capsid antigen in the post-infection culture supernatant was measured using ELISA. The supernatant samples were prepared in triplicate, and the mean capsid antigen level for each drug treatment was further converted to a value relative to mock treatment. Statistically significant differences, determined as in Figure 2D, are indicated on the graphs.
Mentions: The immunofluorescence detection of viral capsid protein offered a qualitative indication of whether a given kinase inhibitor affected the initiation of the infection processes leading to viral gene expression. In order to more quantitatively measure the effect of the drugs on viral propagation, the amount of viral RNA produced in the cells at 24 hpi in the presence or absence of the drugs was measured by quantitative real-time RT-PCR (Figure 3A). Cells treated with genistein, staurosporine, U0126, and LY294002 contained significantly lower amounts of viral RNA than cells treated with the solvent alone, consistent with the finding that these drugs were inhibitory to the expression of viral capsid. Although treatment with wortmannin could show inhibitory effect on viral capsid expression (Figure 1B), it did not translate into a significant effect on viral RNA replication (see Discussion). Not surprisingly, drugs that did not inhibit viral gene expression—inhibitors of MAPK p38s (SB203580), JNK (JNK inhibitor II), Akt (MK2206), and PKA (H89)—had no measurable effect on the extent of viral RNA replication. Treatment with triciribine, NSC23766, or Y27632 induced higher levels of RNA replication and did not inhibit the production of viral RNA. These results support the idea that PI3K activation is important for the initiation of viral infection via a non-Akt, non-Rac mediated pathway.

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