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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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Blebs induction upon ASFV entry in IPAM cells.A) Field Emission SEM of mock-infected and infected cells. Cells were synchronously infected for 10 min (MOI 50) with E70 after serum starved for 24 h. Membrane perturbations are indicated by arrowheads. A magnification of the cell surface detail (boxes) is shown in the lower right panels. Bars: 1 µM. B) After synchronic infection at different times (E70, MOI 50), the cells were fixed and blebs formation (arrowheads) was analyzed by Phase Contrast Microscopy using a 63× objective. C) Blebbistatin treatment inhibits ASFV entry. Cells were treated with DMSO or Blebbistatin 60 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection, at indicated concentrations. After 16 hpi (Ba71V, MOI 1) equivalent amounts of protein were analyzed by immunoblotting with an anti-ASFV antibody. β-actin was detected as a load control. Fold induction was determined by densitometry (mean ±S.D) as shown in the graphic below. D) Rock1 colocalizes with ASFV in blebs (arrows). Cells were infected (Ba71V, MOI 50) and fixed at 30 min after infection. Cells were incubated with, anti-Rock1 (red), anti-p72 (green) and Topro3 (blue) to stain blebs, virus and nuclei, respectively. Images were taken by CLSM and represented as a maximum z-projection of x–y plane and Normasky. Magnifications of the bleb containing Rock1 and viruses (boxes) are shown in the corresponding bottom panels. S.D., standard deviations.
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ppat-1002754-g002: Blebs induction upon ASFV entry in IPAM cells.A) Field Emission SEM of mock-infected and infected cells. Cells were synchronously infected for 10 min (MOI 50) with E70 after serum starved for 24 h. Membrane perturbations are indicated by arrowheads. A magnification of the cell surface detail (boxes) is shown in the lower right panels. Bars: 1 µM. B) After synchronic infection at different times (E70, MOI 50), the cells were fixed and blebs formation (arrowheads) was analyzed by Phase Contrast Microscopy using a 63× objective. C) Blebbistatin treatment inhibits ASFV entry. Cells were treated with DMSO or Blebbistatin 60 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection, at indicated concentrations. After 16 hpi (Ba71V, MOI 1) equivalent amounts of protein were analyzed by immunoblotting with an anti-ASFV antibody. β-actin was detected as a load control. Fold induction was determined by densitometry (mean ±S.D) as shown in the graphic below. D) Rock1 colocalizes with ASFV in blebs (arrows). Cells were infected (Ba71V, MOI 50) and fixed at 30 min after infection. Cells were incubated with, anti-Rock1 (red), anti-p72 (green) and Topro3 (blue) to stain blebs, virus and nuclei, respectively. Images were taken by CLSM and represented as a maximum z-projection of x–y plane and Normasky. Magnifications of the bleb containing Rock1 and viruses (boxes) are shown in the corresponding bottom panels. S.D., standard deviations.

Mentions: To assess whether the ASFV entry also induces membrane perturbation in swine macrophages, the natural target cell of ASFV infection in vivo, the virulent strain E70 was used to synchronously infect IPAM cells at MOI 50. As early as 10 mpi, strong membrane protrusions were observed by FESEM analysis (Figure 2A). To better characterize these membrane rearrangements, IPAM cells were synchronously infected with E70 strain at MOI 50, during 30, 45 and 60 mpi. Next, IPAM cells were fixed and analyzed by optic microscopy. Figure 2B shows images compatible with blebs induced by ASFV infection in swine macrophages from 30 mpi. To prove this point, we achieved an additional experiment showing the inhibition of virus entry with different doses of blebbistatin, an inhibitor of blebbing and macropinocytosis [23], [29], [49]–[51]. Western blot analyses have shown that blebbistatin impairs the entry of the virus in IPAM cells, as the drug inhibits the expression of ASFV proteins when preincubated before virus addition. Hence, when blebbistatin was incubated 60 min after virus addition, a much lower inhibition of viral proteins was observed, thus indicating the role of blebbistatin on early steps of virus entry. Results are presented in Figure 2C.


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)

Blebs induction upon ASFV entry in IPAM cells.A) Field Emission SEM of mock-infected and infected cells. Cells were synchronously infected for 10 min (MOI 50) with E70 after serum starved for 24 h. Membrane perturbations are indicated by arrowheads. A magnification of the cell surface detail (boxes) is shown in the lower right panels. Bars: 1 µM. B) After synchronic infection at different times (E70, MOI 50), the cells were fixed and blebs formation (arrowheads) was analyzed by Phase Contrast Microscopy using a 63× objective. C) Blebbistatin treatment inhibits ASFV entry. Cells were treated with DMSO or Blebbistatin 60 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection, at indicated concentrations. After 16 hpi (Ba71V, MOI 1) equivalent amounts of protein were analyzed by immunoblotting with an anti-ASFV antibody. β-actin was detected as a load control. Fold induction was determined by densitometry (mean ±S.D) as shown in the graphic below. D) Rock1 colocalizes with ASFV in blebs (arrows). Cells were infected (Ba71V, MOI 50) and fixed at 30 min after infection. Cells were incubated with, anti-Rock1 (red), anti-p72 (green) and Topro3 (blue) to stain blebs, virus and nuclei, respectively. Images were taken by CLSM and represented as a maximum z-projection of x–y plane and Normasky. Magnifications of the bleb containing Rock1 and viruses (boxes) are shown in the corresponding bottom panels. S.D., standard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375293&req=5

ppat-1002754-g002: Blebs induction upon ASFV entry in IPAM cells.A) Field Emission SEM of mock-infected and infected cells. Cells were synchronously infected for 10 min (MOI 50) with E70 after serum starved for 24 h. Membrane perturbations are indicated by arrowheads. A magnification of the cell surface detail (boxes) is shown in the lower right panels. Bars: 1 µM. B) After synchronic infection at different times (E70, MOI 50), the cells were fixed and blebs formation (arrowheads) was analyzed by Phase Contrast Microscopy using a 63× objective. C) Blebbistatin treatment inhibits ASFV entry. Cells were treated with DMSO or Blebbistatin 60 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection, at indicated concentrations. After 16 hpi (Ba71V, MOI 1) equivalent amounts of protein were analyzed by immunoblotting with an anti-ASFV antibody. β-actin was detected as a load control. Fold induction was determined by densitometry (mean ±S.D) as shown in the graphic below. D) Rock1 colocalizes with ASFV in blebs (arrows). Cells were infected (Ba71V, MOI 50) and fixed at 30 min after infection. Cells were incubated with, anti-Rock1 (red), anti-p72 (green) and Topro3 (blue) to stain blebs, virus and nuclei, respectively. Images were taken by CLSM and represented as a maximum z-projection of x–y plane and Normasky. Magnifications of the bleb containing Rock1 and viruses (boxes) are shown in the corresponding bottom panels. S.D., standard deviations.
Mentions: To assess whether the ASFV entry also induces membrane perturbation in swine macrophages, the natural target cell of ASFV infection in vivo, the virulent strain E70 was used to synchronously infect IPAM cells at MOI 50. As early as 10 mpi, strong membrane protrusions were observed by FESEM analysis (Figure 2A). To better characterize these membrane rearrangements, IPAM cells were synchronously infected with E70 strain at MOI 50, during 30, 45 and 60 mpi. Next, IPAM cells were fixed and analyzed by optic microscopy. Figure 2B shows images compatible with blebs induced by ASFV infection in swine macrophages from 30 mpi. To prove this point, we achieved an additional experiment showing the inhibition of virus entry with different doses of blebbistatin, an inhibitor of blebbing and macropinocytosis [23], [29], [49]–[51]. Western blot analyses have shown that blebbistatin impairs the entry of the virus in IPAM cells, as the drug inhibits the expression of ASFV proteins when preincubated before virus addition. Hence, when blebbistatin was incubated 60 min after virus addition, a much lower inhibition of viral proteins was observed, thus indicating the role of blebbistatin on early steps of virus entry. Results are presented in Figure 2C.

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