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African swine fever virus uses macropinocytosis to enter host cells.

Sánchez EG, Quintas A, Pérez-Núñez D, Nogal M, Barroso S, Carrascosa ÁL, Revilla Y - PLoS Pathog. (2012)

Bottom Line: Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis.We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis.Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.

ABSTRACT
African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.

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Rac1 plays a critical role in ASFV entry in Vero cells.A–B) Activation of Rac1 during ASFV entry. Vero cells were infected (MOI 10) and 0.Rac1 activation was measured by A) Kit Activation Assay (n = 3; mean ±S.D.) and B) Pak1 PBD-Agarose Beads pull down assay. Fold induction was determined by densitometry (mean ±S.D). C) ASFV infection induces clustering of Rac1. Cells were transfected with pEGFP-Rac1, infected (MOI 10) and stained with Topro3 (blue) and anti-p72 (red). Analyzed images by CLSM were represented as a maximum of z-projection. D–E) Rac1 inhibitor blocks viral entry. Pretreated cells (200 µM Rac1 inhibitor) were infected (MOI 10) for 60 min. D) Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal analyzed by FACS (n = 3, performed in duplicate; mean ±S.D.). E) Cells were incubated with Topro3 (blue), TRITC-phalloidin (red) and anti-p72 (green). Images are represented as a maximum z-projection of x-y plane (bottom panels) and x–z plane (upper panels). F) Expression of inactive form of Rac-1 reduces viral infection. Transfected cells with pcDNA or pGFP-Rac1-N17 were infected (MOI 1) for 16 h. Viral protein synthesis was analyzed by immunoblotting with an anti-p32 antibody. GFP and β-actin levels were measured as a control. G–H) Rac-1 inhibitor affects ASFV infection. G) Viral factory formation was analyzed in pretreated and infected cells (MOI 5) for 16 h. Cells were fixed and stained with Topro3, TRITC-phalloidin and anti-p72. Arrowheads: viral factories. Percentage of the infected cells is represented in left graphic (100 cells/condition; n = 3; mean ±S.D.). H) After 48 hpi (MOI 1) supernatants from treated cells were recovered and lytic viruses were titrated (n = 3). I) ASFV-induced ruffles are inhibited by Rac1 inhibitor. Cells were pretreated (200 µM Rac1 inhibitor), infected (MOI 50) for 10 min, fixed and analyzed by FESEM. S.D., standard deviations.
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ppat-1002754-g006: Rac1 plays a critical role in ASFV entry in Vero cells.A–B) Activation of Rac1 during ASFV entry. Vero cells were infected (MOI 10) and 0.Rac1 activation was measured by A) Kit Activation Assay (n = 3; mean ±S.D.) and B) Pak1 PBD-Agarose Beads pull down assay. Fold induction was determined by densitometry (mean ±S.D). C) ASFV infection induces clustering of Rac1. Cells were transfected with pEGFP-Rac1, infected (MOI 10) and stained with Topro3 (blue) and anti-p72 (red). Analyzed images by CLSM were represented as a maximum of z-projection. D–E) Rac1 inhibitor blocks viral entry. Pretreated cells (200 µM Rac1 inhibitor) were infected (MOI 10) for 60 min. D) Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal analyzed by FACS (n = 3, performed in duplicate; mean ±S.D.). E) Cells were incubated with Topro3 (blue), TRITC-phalloidin (red) and anti-p72 (green). Images are represented as a maximum z-projection of x-y plane (bottom panels) and x–z plane (upper panels). F) Expression of inactive form of Rac-1 reduces viral infection. Transfected cells with pcDNA or pGFP-Rac1-N17 were infected (MOI 1) for 16 h. Viral protein synthesis was analyzed by immunoblotting with an anti-p32 antibody. GFP and β-actin levels were measured as a control. G–H) Rac-1 inhibitor affects ASFV infection. G) Viral factory formation was analyzed in pretreated and infected cells (MOI 5) for 16 h. Cells were fixed and stained with Topro3, TRITC-phalloidin and anti-p72. Arrowheads: viral factories. Percentage of the infected cells is represented in left graphic (100 cells/condition; n = 3; mean ±S.D.). H) After 48 hpi (MOI 1) supernatants from treated cells were recovered and lytic viruses were titrated (n = 3). I) ASFV-induced ruffles are inhibited by Rac1 inhibitor. Cells were pretreated (200 µM Rac1 inhibitor), infected (MOI 50) for 10 min, fixed and analyzed by FESEM. S.D., standard deviations.

Mentions: Since activation of Rac1-GTPase has been involved in the regulation of macropinocytosis by triggering membrane ruffling in the cell [87], we investigated the activation status of Rac1 during the first steps of ASFV entry in Vero cells. Ba71V was used to synchronously infect cells (MOI 10), and Rac1 activation was measured with the G-LISA activation kit following the manufacturer's instructions. The results showed that Rac1 activation is a very fast and strong event during ASFV entry, reaching a maximum (2.5 fold) at 10 mpi compared to mock-infected cells (Figure 6A). It has been shown that Rac1 controls macropinocytosis by interacting with its specific effectors, the p21-activated kinases (Paks), thus modulating actin cytoskeleton dynamics [88], [89]. It is also known that Rac1 binds and activates Pak1 only under its Rac1-GTP active form. To confirm the results obtained by G-LISA, we further analyzed the Rac1 activation during ASFV entry by performing a pull down assay using Pak1-PBD-Agarose Beads, which carried the PBD-Pak1 ready to bind Rac1-GTP. As shown in Figure 6B, Rac1-GTP was found together with the pulled Pak1-PBD-Agarose Beads after 10 min post ASFV infection, slightly diminishing 30 min after the infection. This result further corroborates that ASFV entry induces the formation of the Rac1 active conformation. Since it has been described that Rac1 is contained in blebs and ruffles [22], [23], [90] and, as shown above, ASFV induces these type of the structures when it infects cells, we next analyzed the localization of Rac1 during the process of ASFV entry. To achieve this, Vero cells were first transfected for 24 h with pEGFP-Rac1 (kindly given by Dr. J. Mercer) and then infected with Ba71V, MOI 10. As shown in Figure 6C, we found clusters of the GTPase as early as 10 min after infection. Accordingly with the experiments shown above, this effect was clearly perceptible at 30 mpi, demonstrating, first, that ASFV infection induces accumulation of active Rac1 in ruffling areas, and second, that this is an event that takes place mainly during ASFV entry.


African swine fever virus uses macropinocytosis to enter host cells.

Sánchez EG, Quintas A, Pérez-Núñez D, Nogal M, Barroso S, Carrascosa ÁL, Revilla Y - PLoS Pathog. (2012)

Rac1 plays a critical role in ASFV entry in Vero cells.A–B) Activation of Rac1 during ASFV entry. Vero cells were infected (MOI 10) and 0.Rac1 activation was measured by A) Kit Activation Assay (n = 3; mean ±S.D.) and B) Pak1 PBD-Agarose Beads pull down assay. Fold induction was determined by densitometry (mean ±S.D). C) ASFV infection induces clustering of Rac1. Cells were transfected with pEGFP-Rac1, infected (MOI 10) and stained with Topro3 (blue) and anti-p72 (red). Analyzed images by CLSM were represented as a maximum of z-projection. D–E) Rac1 inhibitor blocks viral entry. Pretreated cells (200 µM Rac1 inhibitor) were infected (MOI 10) for 60 min. D) Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal analyzed by FACS (n = 3, performed in duplicate; mean ±S.D.). E) Cells were incubated with Topro3 (blue), TRITC-phalloidin (red) and anti-p72 (green). Images are represented as a maximum z-projection of x-y plane (bottom panels) and x–z plane (upper panels). F) Expression of inactive form of Rac-1 reduces viral infection. Transfected cells with pcDNA or pGFP-Rac1-N17 were infected (MOI 1) for 16 h. Viral protein synthesis was analyzed by immunoblotting with an anti-p32 antibody. GFP and β-actin levels were measured as a control. G–H) Rac-1 inhibitor affects ASFV infection. G) Viral factory formation was analyzed in pretreated and infected cells (MOI 5) for 16 h. Cells were fixed and stained with Topro3, TRITC-phalloidin and anti-p72. Arrowheads: viral factories. Percentage of the infected cells is represented in left graphic (100 cells/condition; n = 3; mean ±S.D.). H) After 48 hpi (MOI 1) supernatants from treated cells were recovered and lytic viruses were titrated (n = 3). I) ASFV-induced ruffles are inhibited by Rac1 inhibitor. Cells were pretreated (200 µM Rac1 inhibitor), infected (MOI 50) for 10 min, fixed and analyzed by FESEM. S.D., standard deviations.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375293&req=5

ppat-1002754-g006: Rac1 plays a critical role in ASFV entry in Vero cells.A–B) Activation of Rac1 during ASFV entry. Vero cells were infected (MOI 10) and 0.Rac1 activation was measured by A) Kit Activation Assay (n = 3; mean ±S.D.) and B) Pak1 PBD-Agarose Beads pull down assay. Fold induction was determined by densitometry (mean ±S.D). C) ASFV infection induces clustering of Rac1. Cells were transfected with pEGFP-Rac1, infected (MOI 10) and stained with Topro3 (blue) and anti-p72 (red). Analyzed images by CLSM were represented as a maximum of z-projection. D–E) Rac1 inhibitor blocks viral entry. Pretreated cells (200 µM Rac1 inhibitor) were infected (MOI 10) for 60 min. D) Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal analyzed by FACS (n = 3, performed in duplicate; mean ±S.D.). E) Cells were incubated with Topro3 (blue), TRITC-phalloidin (red) and anti-p72 (green). Images are represented as a maximum z-projection of x-y plane (bottom panels) and x–z plane (upper panels). F) Expression of inactive form of Rac-1 reduces viral infection. Transfected cells with pcDNA or pGFP-Rac1-N17 were infected (MOI 1) for 16 h. Viral protein synthesis was analyzed by immunoblotting with an anti-p32 antibody. GFP and β-actin levels were measured as a control. G–H) Rac-1 inhibitor affects ASFV infection. G) Viral factory formation was analyzed in pretreated and infected cells (MOI 5) for 16 h. Cells were fixed and stained with Topro3, TRITC-phalloidin and anti-p72. Arrowheads: viral factories. Percentage of the infected cells is represented in left graphic (100 cells/condition; n = 3; mean ±S.D.). H) After 48 hpi (MOI 1) supernatants from treated cells were recovered and lytic viruses were titrated (n = 3). I) ASFV-induced ruffles are inhibited by Rac1 inhibitor. Cells were pretreated (200 µM Rac1 inhibitor), infected (MOI 50) for 10 min, fixed and analyzed by FESEM. S.D., standard deviations.
Mentions: Since activation of Rac1-GTPase has been involved in the regulation of macropinocytosis by triggering membrane ruffling in the cell [87], we investigated the activation status of Rac1 during the first steps of ASFV entry in Vero cells. Ba71V was used to synchronously infect cells (MOI 10), and Rac1 activation was measured with the G-LISA activation kit following the manufacturer's instructions. The results showed that Rac1 activation is a very fast and strong event during ASFV entry, reaching a maximum (2.5 fold) at 10 mpi compared to mock-infected cells (Figure 6A). It has been shown that Rac1 controls macropinocytosis by interacting with its specific effectors, the p21-activated kinases (Paks), thus modulating actin cytoskeleton dynamics [88], [89]. It is also known that Rac1 binds and activates Pak1 only under its Rac1-GTP active form. To confirm the results obtained by G-LISA, we further analyzed the Rac1 activation during ASFV entry by performing a pull down assay using Pak1-PBD-Agarose Beads, which carried the PBD-Pak1 ready to bind Rac1-GTP. As shown in Figure 6B, Rac1-GTP was found together with the pulled Pak1-PBD-Agarose Beads after 10 min post ASFV infection, slightly diminishing 30 min after the infection. This result further corroborates that ASFV entry induces the formation of the Rac1 active conformation. Since it has been described that Rac1 is contained in blebs and ruffles [22], [23], [90] and, as shown above, ASFV induces these type of the structures when it infects cells, we next analyzed the localization of Rac1 during the process of ASFV entry. To achieve this, Vero cells were first transfected for 24 h with pEGFP-Rac1 (kindly given by Dr. J. Mercer) and then infected with Ba71V, MOI 10. As shown in Figure 6C, we found clusters of the GTPase as early as 10 min after infection. Accordingly with the experiments shown above, this effect was clearly perceptible at 30 mpi, demonstrating, first, that ASFV infection induces accumulation of active Rac1 in ruffling areas, and second, that this is an event that takes place mainly during ASFV entry.

Bottom Line: Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis.We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis.Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.

ABSTRACT
African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.

Show MeSH
Related in: MedlinePlus