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Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane.

Azab W, Gramatica A, Herrmann A, Osterrieder N - MBio (2015)

Bottom Line: We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca(2+) levels.EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca(2+) release, while EHV-4 with gH1 triggered significant Ca(2+) release.These viruses have developed a number of strategies for successful entry into different cell types.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Berlin, Germany wfazab@zedat.fu-berlin.de.

No MeSH data available.


Related in: MedlinePlus

EHV-1 gH and cellular α4β1-integrin mediate cytosolic Ca2+ increase during virus entry. (A) ED cells were loaded with the Ca2+ indicator Fura-2AM and exposed to EHV-1, EHV-4, EHV-1gH4, or EHV-4gH1. (B) ED cells were incubated with 20 µg/ml of anti–α4β1-integrin MAb P4C2 for 1 h at 37°C. After washing, cells were loaded with Fura-2AM and exposed to EHV-1 and EHV-4gH1. (C) EHV-1 and EHV-4gH1 were incubated with soluble α4β1-integrin for 1 h at 37°C. The cells were loaded with Fura-2AM and exposed to the viruses. Changes in cytosolic Ca2+ levels were monitored using epifluorescence microscopy. Viruses were added at 50-s time point. The average from three independent experiments of fluorescence intensities of Fura-2AM versus time of exposure of ED cells to the viruses is shown. (A) P < 0.001 indicates a significant difference between EHV-1gH4 and EHV-4gH1 compared to parental EHV-1 and EHV-4, respectively. (B) P < 0.01 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of the integrin antibodies. (C) P < 0.001 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of soluble α4β1-integrin (Sol intg).
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fig2: EHV-1 gH and cellular α4β1-integrin mediate cytosolic Ca2+ increase during virus entry. (A) ED cells were loaded with the Ca2+ indicator Fura-2AM and exposed to EHV-1, EHV-4, EHV-1gH4, or EHV-4gH1. (B) ED cells were incubated with 20 µg/ml of anti–α4β1-integrin MAb P4C2 for 1 h at 37°C. After washing, cells were loaded with Fura-2AM and exposed to EHV-1 and EHV-4gH1. (C) EHV-1 and EHV-4gH1 were incubated with soluble α4β1-integrin for 1 h at 37°C. The cells were loaded with Fura-2AM and exposed to the viruses. Changes in cytosolic Ca2+ levels were monitored using epifluorescence microscopy. Viruses were added at 50-s time point. The average from three independent experiments of fluorescence intensities of Fura-2AM versus time of exposure of ED cells to the viruses is shown. (A) P < 0.001 indicates a significant difference between EHV-1gH4 and EHV-4gH1 compared to parental EHV-1 and EHV-4, respectively. (B) P < 0.01 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of the integrin antibodies. (C) P < 0.001 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of soluble α4β1-integrin (Sol intg).

Mentions: In a previous study, we showed that viral gH and cellular α4β1-integrins play an important role during EHV-1 and EHV-4 entry and that both can act as a routing factor capable of altering the entry pathways of the viruses (22). Following up on these results, we explored the possibility that interaction between gH and α4β1-integrins may trigger release of Ca2+ from intracellular stores. Based on live-cell-imaging measurements, we concluded that, in contrast to parental EHV-1, EHV-1 bearing gH4 (EHV-1gH4) was unable to trigger an increase of cytosolic Ca2+. On the other hand, EHV-4 expressing and having in its envelope gH1 (EHV-4gH1) induced a significant increase of cytosolic Ca2+ (Fig. 2A). It is important to note that only EHV-1 gH has an integrin-binding motif, serine-aspartate-isoleucine (SDI), that mediates binding to α4β1-integrins. EHV-4 gH specifies an alanine-aspartate-isoleucine (ADI) motif that cannot mediate integrin binding (39). Addition of EHV-1gHS440A, an EHV-1 mutant in which the α4β1-integrin binding motif SDI was mutated to ADI, had no effect on cytosolic Ca2+ levels (see Movie S3 in the supplemental material). From the experiments, we concluded that gH plays a role in increasing cytosolic Ca2+ levels through a mechanism that is dependent on the interaction of gH with integrins expressed on the cell surface.


Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane.

Azab W, Gramatica A, Herrmann A, Osterrieder N - MBio (2015)

EHV-1 gH and cellular α4β1-integrin mediate cytosolic Ca2+ increase during virus entry. (A) ED cells were loaded with the Ca2+ indicator Fura-2AM and exposed to EHV-1, EHV-4, EHV-1gH4, or EHV-4gH1. (B) ED cells were incubated with 20 µg/ml of anti–α4β1-integrin MAb P4C2 for 1 h at 37°C. After washing, cells were loaded with Fura-2AM and exposed to EHV-1 and EHV-4gH1. (C) EHV-1 and EHV-4gH1 were incubated with soluble α4β1-integrin for 1 h at 37°C. The cells were loaded with Fura-2AM and exposed to the viruses. Changes in cytosolic Ca2+ levels were monitored using epifluorescence microscopy. Viruses were added at 50-s time point. The average from three independent experiments of fluorescence intensities of Fura-2AM versus time of exposure of ED cells to the viruses is shown. (A) P < 0.001 indicates a significant difference between EHV-1gH4 and EHV-4gH1 compared to parental EHV-1 and EHV-4, respectively. (B) P < 0.01 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of the integrin antibodies. (C) P < 0.001 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of soluble α4β1-integrin (Sol intg).
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fig2: EHV-1 gH and cellular α4β1-integrin mediate cytosolic Ca2+ increase during virus entry. (A) ED cells were loaded with the Ca2+ indicator Fura-2AM and exposed to EHV-1, EHV-4, EHV-1gH4, or EHV-4gH1. (B) ED cells were incubated with 20 µg/ml of anti–α4β1-integrin MAb P4C2 for 1 h at 37°C. After washing, cells were loaded with Fura-2AM and exposed to EHV-1 and EHV-4gH1. (C) EHV-1 and EHV-4gH1 were incubated with soluble α4β1-integrin for 1 h at 37°C. The cells were loaded with Fura-2AM and exposed to the viruses. Changes in cytosolic Ca2+ levels were monitored using epifluorescence microscopy. Viruses were added at 50-s time point. The average from three independent experiments of fluorescence intensities of Fura-2AM versus time of exposure of ED cells to the viruses is shown. (A) P < 0.001 indicates a significant difference between EHV-1gH4 and EHV-4gH1 compared to parental EHV-1 and EHV-4, respectively. (B) P < 0.01 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of the integrin antibodies. (C) P < 0.001 indicates a significant difference between EHV-1 and EHV-4gH1 viruses in the presence or absence of soluble α4β1-integrin (Sol intg).
Mentions: In a previous study, we showed that viral gH and cellular α4β1-integrins play an important role during EHV-1 and EHV-4 entry and that both can act as a routing factor capable of altering the entry pathways of the viruses (22). Following up on these results, we explored the possibility that interaction between gH and α4β1-integrins may trigger release of Ca2+ from intracellular stores. Based on live-cell-imaging measurements, we concluded that, in contrast to parental EHV-1, EHV-1 bearing gH4 (EHV-1gH4) was unable to trigger an increase of cytosolic Ca2+. On the other hand, EHV-4 expressing and having in its envelope gH1 (EHV-4gH1) induced a significant increase of cytosolic Ca2+ (Fig. 2A). It is important to note that only EHV-1 gH has an integrin-binding motif, serine-aspartate-isoleucine (SDI), that mediates binding to α4β1-integrins. EHV-4 gH specifies an alanine-aspartate-isoleucine (ADI) motif that cannot mediate integrin binding (39). Addition of EHV-1gHS440A, an EHV-1 mutant in which the α4β1-integrin binding motif SDI was mutated to ADI, had no effect on cytosolic Ca2+ levels (see Movie S3 in the supplemental material). From the experiments, we concluded that gH plays a role in increasing cytosolic Ca2+ levels through a mechanism that is dependent on the interaction of gH with integrins expressed on the cell surface.

Bottom Line: We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca(2+) levels.EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca(2+) release, while EHV-4 with gH1 triggered significant Ca(2+) release.These viruses have developed a number of strategies for successful entry into different cell types.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Berlin, Germany wfazab@zedat.fu-berlin.de.

No MeSH data available.


Related in: MedlinePlus