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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

Importance of timing of kinase inhibition during HAstV1 infection. (A)Time course of activation of MAPKs and Akt. Lysate was prepared from either mock-infected (Mock) or HAstV1-infected (Infected) Caco-2 cells at designated time points. The lysates were subjected to Western blot to detect either total kinases (ERK, p38, JNK, and Akt) or their phosphorylated forms [(pERK, pp38, pJNK, and pAkt) MAPKs and Akt]. The two bands in the “ERK” and “pERK” panels represent ERK1 (44 kDa) and ERK2 (42 kDa), respectively. The two bands in “JNK” and “pJNK” panels represent JNK1 (54 kDa) and JNK2 (46 kDa), respectively. (B)Effect of inhibitors on kinase activation. Lysates prepared at designated time points from HAstV1-infected Caco-2 cells in the presence of solvent alone (DMSO), LY294002, or U0126 were subjected to Western blot for either total ERK (ERK) or its phosphorylated form (pERK). ERK1 and 2 bands are indicated by an arrowhead and an arrow, respectively. (C)Quantitative presentation of the Western blot signal shown in A. Digital images of the bands were quantitated using ImageJ. The vertical line indicates the relative value of the signal intensity divided by the value of the band for each mock-infected sample, at 0.25 hpi, in each experimental group. The black and gray bars represent values for the mock- and HAstV1-infected sample, respectively. Note that, due to large differences, the scale of the vertical bar representing “Akt” differs from that of others. (D)Effects of inhibitors on capsid protein expression. Caco-2 cells were infected and examined by immunofluorescence analysis as in Figure 1, except that solvent alone or inhibitors were added at indicated time points and maintained until fixing at 24 hpi. For each time point, the proportions of cells positive for capsid protein expression were examined statistically as described in Methods. (*P < 0.05; **P < 0.001).
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Figure 2: Importance of timing of kinase inhibition during HAstV1 infection. (A)Time course of activation of MAPKs and Akt. Lysate was prepared from either mock-infected (Mock) or HAstV1-infected (Infected) Caco-2 cells at designated time points. The lysates were subjected to Western blot to detect either total kinases (ERK, p38, JNK, and Akt) or their phosphorylated forms [(pERK, pp38, pJNK, and pAkt) MAPKs and Akt]. The two bands in the “ERK” and “pERK” panels represent ERK1 (44 kDa) and ERK2 (42 kDa), respectively. The two bands in “JNK” and “pJNK” panels represent JNK1 (54 kDa) and JNK2 (46 kDa), respectively. (B)Effect of inhibitors on kinase activation. Lysates prepared at designated time points from HAstV1-infected Caco-2 cells in the presence of solvent alone (DMSO), LY294002, or U0126 were subjected to Western blot for either total ERK (ERK) or its phosphorylated form (pERK). ERK1 and 2 bands are indicated by an arrowhead and an arrow, respectively. (C)Quantitative presentation of the Western blot signal shown in A. Digital images of the bands were quantitated using ImageJ. The vertical line indicates the relative value of the signal intensity divided by the value of the band for each mock-infected sample, at 0.25 hpi, in each experimental group. The black and gray bars represent values for the mock- and HAstV1-infected sample, respectively. Note that, due to large differences, the scale of the vertical bar representing “Akt” differs from that of others. (D)Effects of inhibitors on capsid protein expression. Caco-2 cells were infected and examined by immunofluorescence analysis as in Figure 1, except that solvent alone or inhibitors were added at indicated time points and maintained until fixing at 24 hpi. For each time point, the proportions of cells positive for capsid protein expression were examined statistically as described in Methods. (*P < 0.05; **P < 0.001).

Mentions: We were also able to confirm that ERK1/2 activation occurs at an early stage of HAstV1 infection. The phosphorylation level of various kinases was examined at different times post-infection by Western blotting for both phosphorylated and phosphorylation-independent epitopes of each kinase (Figure 2A). The signal intensity of each band relative to that of each mock-infected sample at 0.25 hpi is presented in Figure 2C. Compared with that of the mock-infected sample, the phosphorylation levels of ERK1/2 were noticeably elevated at the early time points (0.25 hpi and 0.5 hpi) (Figure 2A, “pERK” and Figure 2C, “ERK”). Similarly, the p38 phosphorylation level appeared to be elevated at 0.25 hpi (Figure 2A, “pp38” and Figure 2C, “p38”). A marginal increase in the phosphorylation level of JNK was observed in the infected cells throughout the time points examined (Figure 2A, “pJNK” and Figure 2C, “JNK”). However, only the phosphorylation of ERK1/2, and not that of p38 and JNK, was necessary for infection, judged from the results of the capsid protein expression assay performed with inhibitors specific to these kinases (Figure 1A). We noted that the level of phosphorylated ERK1/2 increased at 8 hpi (Figure 2A, right “pERK”), an observation not reported earlier [19]. This is unlikely to be related to any infection event because phosphorylated ERK1/2 was similarly elevated at this time point in the mock-infected sample (left “pERK” panel).


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)

Importance of timing of kinase inhibition during HAstV1 infection. (A)Time course of activation of MAPKs and Akt. Lysate was prepared from either mock-infected (Mock) or HAstV1-infected (Infected) Caco-2 cells at designated time points. The lysates were subjected to Western blot to detect either total kinases (ERK, p38, JNK, and Akt) or their phosphorylated forms [(pERK, pp38, pJNK, and pAkt) MAPKs and Akt]. The two bands in the “ERK” and “pERK” panels represent ERK1 (44 kDa) and ERK2 (42 kDa), respectively. The two bands in “JNK” and “pJNK” panels represent JNK1 (54 kDa) and JNK2 (46 kDa), respectively. (B)Effect of inhibitors on kinase activation. Lysates prepared at designated time points from HAstV1-infected Caco-2 cells in the presence of solvent alone (DMSO), LY294002, or U0126 were subjected to Western blot for either total ERK (ERK) or its phosphorylated form (pERK). ERK1 and 2 bands are indicated by an arrowhead and an arrow, respectively. (C)Quantitative presentation of the Western blot signal shown in A. Digital images of the bands were quantitated using ImageJ. The vertical line indicates the relative value of the signal intensity divided by the value of the band for each mock-infected sample, at 0.25 hpi, in each experimental group. The black and gray bars represent values for the mock- and HAstV1-infected sample, respectively. Note that, due to large differences, the scale of the vertical bar representing “Akt” differs from that of others. (D)Effects of inhibitors on capsid protein expression. Caco-2 cells were infected and examined by immunofluorescence analysis as in Figure 1, except that solvent alone or inhibitors were added at indicated time points and maintained until fixing at 24 hpi. For each time point, the proportions of cells positive for capsid protein expression were examined statistically as described in Methods. (*P < 0.05; **P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Importance of timing of kinase inhibition during HAstV1 infection. (A)Time course of activation of MAPKs and Akt. Lysate was prepared from either mock-infected (Mock) or HAstV1-infected (Infected) Caco-2 cells at designated time points. The lysates were subjected to Western blot to detect either total kinases (ERK, p38, JNK, and Akt) or their phosphorylated forms [(pERK, pp38, pJNK, and pAkt) MAPKs and Akt]. The two bands in the “ERK” and “pERK” panels represent ERK1 (44 kDa) and ERK2 (42 kDa), respectively. The two bands in “JNK” and “pJNK” panels represent JNK1 (54 kDa) and JNK2 (46 kDa), respectively. (B)Effect of inhibitors on kinase activation. Lysates prepared at designated time points from HAstV1-infected Caco-2 cells in the presence of solvent alone (DMSO), LY294002, or U0126 were subjected to Western blot for either total ERK (ERK) or its phosphorylated form (pERK). ERK1 and 2 bands are indicated by an arrowhead and an arrow, respectively. (C)Quantitative presentation of the Western blot signal shown in A. Digital images of the bands were quantitated using ImageJ. The vertical line indicates the relative value of the signal intensity divided by the value of the band for each mock-infected sample, at 0.25 hpi, in each experimental group. The black and gray bars represent values for the mock- and HAstV1-infected sample, respectively. Note that, due to large differences, the scale of the vertical bar representing “Akt” differs from that of others. (D)Effects of inhibitors on capsid protein expression. Caco-2 cells were infected and examined by immunofluorescence analysis as in Figure 1, except that solvent alone or inhibitors were added at indicated time points and maintained until fixing at 24 hpi. For each time point, the proportions of cells positive for capsid protein expression were examined statistically as described in Methods. (*P < 0.05; **P < 0.001).
Mentions: We were also able to confirm that ERK1/2 activation occurs at an early stage of HAstV1 infection. The phosphorylation level of various kinases was examined at different times post-infection by Western blotting for both phosphorylated and phosphorylation-independent epitopes of each kinase (Figure 2A). The signal intensity of each band relative to that of each mock-infected sample at 0.25 hpi is presented in Figure 2C. Compared with that of the mock-infected sample, the phosphorylation levels of ERK1/2 were noticeably elevated at the early time points (0.25 hpi and 0.5 hpi) (Figure 2A, “pERK” and Figure 2C, “ERK”). Similarly, the p38 phosphorylation level appeared to be elevated at 0.25 hpi (Figure 2A, “pp38” and Figure 2C, “p38”). A marginal increase in the phosphorylation level of JNK was observed in the infected cells throughout the time points examined (Figure 2A, “pJNK” and Figure 2C, “JNK”). However, only the phosphorylation of ERK1/2, and not that of p38 and JNK, was necessary for infection, judged from the results of the capsid protein expression assay performed with inhibitors specific to these kinases (Figure 1A). We noted that the level of phosphorylated ERK1/2 increased at 8 hpi (Figure 2A, right “pERK”), an observation not reported earlier [19]. This is unlikely to be related to any infection event because phosphorylated ERK1/2 was similarly elevated at this time point in the mock-infected sample (left “pERK” panel).

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