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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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Pak1 is required for ASFV entry in Vero cells.A) ASFV activates Pak1 at early times post infection. Cells were infected (MOI 5) and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot. Levels of total Pak1 were measured as a control. Fold induction was determined by densitometry (mean ±S.D). B–D) IPA-3 inhibits ASFV entry. B) Cells were pretreated with DMSO or 30 µM IPA-3 and infected (MOI 10) for 60 min to analyze ASFV uptake by FACS. The graph shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 9, performed in duplicate; mean ±S.D.). C) Viral protein synthesis was analyzed in infected cells (MOI 1) at 16 hpi in the presence of IPA-3 at the indicated concentrations. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. D) Supernatants from DMSO or 5 µM IPA-3 treated cells after 48 hpi (MOI 1) were recovered. Lytic viruses were titrated in Vero monolayers and plotted in the table (n = 3). E–F) Pak1 mutant reduces ASFV infection. E) Vero cells were transfected with pEGFP-Pak1-WT, pEGFP-Pak1-AID (Pak D/N form) and pEGFP-Pak1-T423E (Pak C/A form) for 24 h. Then, cells were infected (MOI 1) for 16 h and viral protein synthesis was analyzed by immunoblotting with an anti-ASFV antibody. GFP expression was measured as a control of transfection. β-actin was detected as a load control. F) Fold induction was determined by densitometry and represented in the graphic (mean ±S.D). S.D., standard deviation. * Unspecific cellular protein detected by the antibody.
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ppat-1002754-g007: Pak1 is required for ASFV entry in Vero cells.A) ASFV activates Pak1 at early times post infection. Cells were infected (MOI 5) and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot. Levels of total Pak1 were measured as a control. Fold induction was determined by densitometry (mean ±S.D). B–D) IPA-3 inhibits ASFV entry. B) Cells were pretreated with DMSO or 30 µM IPA-3 and infected (MOI 10) for 60 min to analyze ASFV uptake by FACS. The graph shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 9, performed in duplicate; mean ±S.D.). C) Viral protein synthesis was analyzed in infected cells (MOI 1) at 16 hpi in the presence of IPA-3 at the indicated concentrations. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. D) Supernatants from DMSO or 5 µM IPA-3 treated cells after 48 hpi (MOI 1) were recovered. Lytic viruses were titrated in Vero monolayers and plotted in the table (n = 3). E–F) Pak1 mutant reduces ASFV infection. E) Vero cells were transfected with pEGFP-Pak1-WT, pEGFP-Pak1-AID (Pak D/N form) and pEGFP-Pak1-T423E (Pak C/A form) for 24 h. Then, cells were infected (MOI 1) for 16 h and viral protein synthesis was analyzed by immunoblotting with an anti-ASFV antibody. GFP expression was measured as a control of transfection. β-actin was detected as a load control. F) Fold induction was determined by densitometry and represented in the graphic (mean ±S.D). S.D., standard deviation. * Unspecific cellular protein detected by the antibody.

Mentions: To determine whether Pak1 was activated during ASFV entry, we first analyzed the phosphorylation on Thr423 in Vero cells synchronously infected (MOI 5) with Ba71V. At different times post infection, samples were collected and analyzed by immunoblotting using an anti-phospho-Pak1 Thr423 antibody. As early as 30 mpi, phosphorylation of Pak1 could be detected, increasing until 120 mpi (Figure 7A).


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)

Pak1 is required for ASFV entry in Vero cells.A) ASFV activates Pak1 at early times post infection. Cells were infected (MOI 5) and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot. Levels of total Pak1 were measured as a control. Fold induction was determined by densitometry (mean ±S.D). B–D) IPA-3 inhibits ASFV entry. B) Cells were pretreated with DMSO or 30 µM IPA-3 and infected (MOI 10) for 60 min to analyze ASFV uptake by FACS. The graph shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 9, performed in duplicate; mean ±S.D.). C) Viral protein synthesis was analyzed in infected cells (MOI 1) at 16 hpi in the presence of IPA-3 at the indicated concentrations. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. D) Supernatants from DMSO or 5 µM IPA-3 treated cells after 48 hpi (MOI 1) were recovered. Lytic viruses were titrated in Vero monolayers and plotted in the table (n = 3). E–F) Pak1 mutant reduces ASFV infection. E) Vero cells were transfected with pEGFP-Pak1-WT, pEGFP-Pak1-AID (Pak D/N form) and pEGFP-Pak1-T423E (Pak C/A form) for 24 h. Then, cells were infected (MOI 1) for 16 h and viral protein synthesis was analyzed by immunoblotting with an anti-ASFV antibody. GFP expression was measured as a control of transfection. β-actin was detected as a load control. F) Fold induction was determined by densitometry and represented in the graphic (mean ±S.D). S.D., standard deviation. * Unspecific cellular protein detected by the antibody.
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Related In: Results  -  Collection

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ppat-1002754-g007: Pak1 is required for ASFV entry in Vero cells.A) ASFV activates Pak1 at early times post infection. Cells were infected (MOI 5) and phosphorylation of Pak1 (Thr423) was determined at different times after infection by Western blot. Levels of total Pak1 were measured as a control. Fold induction was determined by densitometry (mean ±S.D). B–D) IPA-3 inhibits ASFV entry. B) Cells were pretreated with DMSO or 30 µM IPA-3 and infected (MOI 10) for 60 min to analyze ASFV uptake by FACS. The graph shows percentage of virus entry relative to DMSO control, measured as p72 signal (n = 9, performed in duplicate; mean ±S.D.). C) Viral protein synthesis was analyzed in infected cells (MOI 1) at 16 hpi in the presence of IPA-3 at the indicated concentrations. Equivalent amounts of protein were analyzed by Western blot with an anti-ASFV antibody. D) Supernatants from DMSO or 5 µM IPA-3 treated cells after 48 hpi (MOI 1) were recovered. Lytic viruses were titrated in Vero monolayers and plotted in the table (n = 3). E–F) Pak1 mutant reduces ASFV infection. E) Vero cells were transfected with pEGFP-Pak1-WT, pEGFP-Pak1-AID (Pak D/N form) and pEGFP-Pak1-T423E (Pak C/A form) for 24 h. Then, cells were infected (MOI 1) for 16 h and viral protein synthesis was analyzed by immunoblotting with an anti-ASFV antibody. GFP expression was measured as a control of transfection. β-actin was detected as a load control. F) Fold induction was determined by densitometry and represented in the graphic (mean ±S.D). S.D., standard deviation. * Unspecific cellular protein detected by the antibody.
Mentions: To determine whether Pak1 was activated during ASFV entry, we first analyzed the phosphorylation on Thr423 in Vero cells synchronously infected (MOI 5) with Ba71V. At different times post infection, samples were collected and analyzed by immunoblotting using an anti-phospho-Pak1 Thr423 antibody. As early as 30 mpi, phosphorylation of Pak1 could be detected, increasing until 120 mpi (Figure 7A).

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