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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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Actin dynamics is important for first steps during ASFV entry in Vero cells.A–D) Disruption of actin dynamics reduces the entry of ASFV. A) Uptake assays were performed by FACS. Pretreated cells with DMSO or 8 µM Cyto D were infected (MOI 10) for 60 min. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 3, performed in duplicate; mean ±S.D). B) Cells were pretreated (4 µM Cyto D) and infected (MOI 1) for 16 h. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. β-actin was detected as a load control. C) After 48 hpi (MOI 1) supernatants from treated cells (8 µM Cyto D) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). D) Development of viral factories (arrowheads) was analyzed by CLSM after treatment (8 µM Cyto D) and infected (MOI 5) for 16 h. Fixed cells were stained with Topro3 (blue), TRITC-phalloidin (red), and anti-p72 (green) to visualize cell nuclei, actin filaments and viral factories, respectively. Images of a mid z-section are shown. The percentage of infected cells of three independent experiments from CLSM images (100 cells per condition) is represented in graphic format (mean ±S.D.). E–F) ASFV infection induces rearrangements of the actin cytoskeleton. Cells were infected at a MOI of 50 pfu/cell (E) or transfected with pEGFP-actin for 16 h and then infected (MOI 50). For both, E and F, cells were fixed at indicated times post infection and incubated with Alexa Fluor 488-phalloidin (E), anti-p72 and Topro3 (E and F) to stain actin filaments, viral particles and cell nuclei, respectively. Z-slides images were taken by CLSM and represented as a maximum of z-projection. S.D., standard deviation; Cyto D, Cytochalasin D. * Unspecific cellular protein detected by the antibody.
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ppat-1002754-g004: Actin dynamics is important for first steps during ASFV entry in Vero cells.A–D) Disruption of actin dynamics reduces the entry of ASFV. A) Uptake assays were performed by FACS. Pretreated cells with DMSO or 8 µM Cyto D were infected (MOI 10) for 60 min. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 3, performed in duplicate; mean ±S.D). B) Cells were pretreated (4 µM Cyto D) and infected (MOI 1) for 16 h. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. β-actin was detected as a load control. C) After 48 hpi (MOI 1) supernatants from treated cells (8 µM Cyto D) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). D) Development of viral factories (arrowheads) was analyzed by CLSM after treatment (8 µM Cyto D) and infected (MOI 5) for 16 h. Fixed cells were stained with Topro3 (blue), TRITC-phalloidin (red), and anti-p72 (green) to visualize cell nuclei, actin filaments and viral factories, respectively. Images of a mid z-section are shown. The percentage of infected cells of three independent experiments from CLSM images (100 cells per condition) is represented in graphic format (mean ±S.D.). E–F) ASFV infection induces rearrangements of the actin cytoskeleton. Cells were infected at a MOI of 50 pfu/cell (E) or transfected with pEGFP-actin for 16 h and then infected (MOI 50). For both, E and F, cells were fixed at indicated times post infection and incubated with Alexa Fluor 488-phalloidin (E), anti-p72 and Topro3 (E and F) to stain actin filaments, viral particles and cell nuclei, respectively. Z-slides images were taken by CLSM and represented as a maximum of z-projection. S.D., standard deviation; Cyto D, Cytochalasin D. * Unspecific cellular protein detected by the antibody.

Mentions: To demonstrate whether ASFV depends on actin to enter cells, we used Cyto D, which binds to the positive end of F-actin impairing further addition of G-actin, thus preventing growth of the microfilament [67]. Vero cells were pretreated with Cyto D at a concentration of 8 µM and ASFV uptake (MOI 10) at 60 mpi was next analyzed by FACS. As shown in Figure 4A, the disruption of actin dynamics by the inhibitor reduced ASFV entry in a percentage of about 50%. To assess whether the drug impairs the synthesis of viral proteins, Vero cells were untreated or treated with Cyto D (4 µM) and then infected with Ba71V, MOI 1. After 16 hpi, we used a specific antiserum against both early and late ASFV proteins (generated in our lab), to analyze viral protein expression. As expected, Cyto D treatment importantly reduced both the synthesis of p32, one of the main ASFV early proteins, and the synthesis of p12, p17 and p72, three typical late proteins in the ASFV cycle (Figure 4B). In agreement with this, both virus production and viral factories clearly diminished as shown in Figure 4C and 4D, respectively. However, it is noteworthy that even in the presence of Cyto D, a number of virions seem able to enter the cell and induce a productive infection, thus suggesting that the actin cytoskeleton is involved in ASFV entry and also in successive post-entry steps, as shown in Figure S4B.


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)

Actin dynamics is important for first steps during ASFV entry in Vero cells.A–D) Disruption of actin dynamics reduces the entry of ASFV. A) Uptake assays were performed by FACS. Pretreated cells with DMSO or 8 µM Cyto D were infected (MOI 10) for 60 min. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 3, performed in duplicate; mean ±S.D). B) Cells were pretreated (4 µM Cyto D) and infected (MOI 1) for 16 h. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. β-actin was detected as a load control. C) After 48 hpi (MOI 1) supernatants from treated cells (8 µM Cyto D) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). D) Development of viral factories (arrowheads) was analyzed by CLSM after treatment (8 µM Cyto D) and infected (MOI 5) for 16 h. Fixed cells were stained with Topro3 (blue), TRITC-phalloidin (red), and anti-p72 (green) to visualize cell nuclei, actin filaments and viral factories, respectively. Images of a mid z-section are shown. The percentage of infected cells of three independent experiments from CLSM images (100 cells per condition) is represented in graphic format (mean ±S.D.). E–F) ASFV infection induces rearrangements of the actin cytoskeleton. Cells were infected at a MOI of 50 pfu/cell (E) or transfected with pEGFP-actin for 16 h and then infected (MOI 50). For both, E and F, cells were fixed at indicated times post infection and incubated with Alexa Fluor 488-phalloidin (E), anti-p72 and Topro3 (E and F) to stain actin filaments, viral particles and cell nuclei, respectively. Z-slides images were taken by CLSM and represented as a maximum of z-projection. S.D., standard deviation; Cyto D, Cytochalasin D. * Unspecific cellular protein detected by the antibody.
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Related In: Results  -  Collection

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

ppat-1002754-g004: Actin dynamics is important for first steps during ASFV entry in Vero cells.A–D) Disruption of actin dynamics reduces the entry of ASFV. A) Uptake assays were performed by FACS. Pretreated cells with DMSO or 8 µM Cyto D were infected (MOI 10) for 60 min. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 3, performed in duplicate; mean ±S.D). B) Cells were pretreated (4 µM Cyto D) and infected (MOI 1) for 16 h. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. β-actin was detected as a load control. C) After 48 hpi (MOI 1) supernatants from treated cells (8 µM Cyto D) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). D) Development of viral factories (arrowheads) was analyzed by CLSM after treatment (8 µM Cyto D) and infected (MOI 5) for 16 h. Fixed cells were stained with Topro3 (blue), TRITC-phalloidin (red), and anti-p72 (green) to visualize cell nuclei, actin filaments and viral factories, respectively. Images of a mid z-section are shown. The percentage of infected cells of three independent experiments from CLSM images (100 cells per condition) is represented in graphic format (mean ±S.D.). E–F) ASFV infection induces rearrangements of the actin cytoskeleton. Cells were infected at a MOI of 50 pfu/cell (E) or transfected with pEGFP-actin for 16 h and then infected (MOI 50). For both, E and F, cells were fixed at indicated times post infection and incubated with Alexa Fluor 488-phalloidin (E), anti-p72 and Topro3 (E and F) to stain actin filaments, viral particles and cell nuclei, respectively. Z-slides images were taken by CLSM and represented as a maximum of z-projection. S.D., standard deviation; Cyto D, Cytochalasin D. * Unspecific cellular protein detected by the antibody.
Mentions: To demonstrate whether ASFV depends on actin to enter cells, we used Cyto D, which binds to the positive end of F-actin impairing further addition of G-actin, thus preventing growth of the microfilament [67]. Vero cells were pretreated with Cyto D at a concentration of 8 µM and ASFV uptake (MOI 10) at 60 mpi was next analyzed by FACS. As shown in Figure 4A, the disruption of actin dynamics by the inhibitor reduced ASFV entry in a percentage of about 50%. To assess whether the drug impairs the synthesis of viral proteins, Vero cells were untreated or treated with Cyto D (4 µM) and then infected with Ba71V, MOI 1. After 16 hpi, we used a specific antiserum against both early and late ASFV proteins (generated in our lab), to analyze viral protein expression. As expected, Cyto D treatment importantly reduced both the synthesis of p32, one of the main ASFV early proteins, and the synthesis of p12, p17 and p72, three typical late proteins in the ASFV cycle (Figure 4B). In agreement with this, both virus production and viral factories clearly diminished as shown in Figure 4C and 4D, respectively. However, it is noteworthy that even in the presence of Cyto D, a number of virions seem able to enter the cell and induce a productive infection, thus suggesting that the actin cytoskeleton is involved in ASFV entry and also in successive post-entry steps, as shown in Figure S4B.

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