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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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EIPA treatment inhibits ASFV entry and infection in Vero cells.A–B) EIPA inhibits ASFV uptake. Cells were pretreated (DMSO or EIPA) and infected (MOI 10) for 60 min. A) Infected cells were analyzed by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal. (n = 7, performed in duplicate; mean±S.D.) B) Cells were incubated with Topro3, TRITC-phalloidin and anti-p72 to stain nuclei (blue), actin filaments (red) and viral particles (green), respectively. Images were taken by CLSM and represented as a maximum z- projection of x–y plane (bottom panels) and x–z plane (upper panels). C–E) The infection is inhibited by EIPA. C) Pretreated cells (20 µM EIPA) were infected (MOI 1) for 16 h and analyzed by immunoblotting with an anti-p72 and an anti-ASFV polyclonal antibodies. D) Pretreated cells (60 µM EIPA) were infected (MOI 5) and stained with Topro3, TRITC-phalloidin and anti-p72. Images were taken by CLSM (mid z-section). Arrowheads: viral factories. E) Supernatants from pretreated (20 µM EIPA) and infected cells (MOI 1) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). F) Bypass of EIPA of ASFV infectivity. Acid mediated bypass was performed and samples of pretreated (20 µM EIPA) and infected cells (MOI 1) for 16 h were analyzed by immunoblotting with an anti-ASFV antibody. G–H) ASFV colocalizes with dextran and induces its uptake. G) Cells were pretreated (60 µM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with 647-dextran for 15 min and analyzed by FACS (n = 3; mean ±S.D.). H) After 30 mpi cells were pulsed with Texas-red-dextran for 15 min and incubated with anti-p72 antibody. Images were taken by CLSM (mid z-section) and Nomarsky. Arrowheads: dextran-virus colocalization. β-actin: load control. S.D., standard deviations. * Unspecific cellular protein detected by the antibody.
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ppat-1002754-g003: EIPA treatment inhibits ASFV entry and infection in Vero cells.A–B) EIPA inhibits ASFV uptake. Cells were pretreated (DMSO or EIPA) and infected (MOI 10) for 60 min. A) Infected cells were analyzed by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal. (n = 7, performed in duplicate; mean±S.D.) B) Cells were incubated with Topro3, TRITC-phalloidin and anti-p72 to stain nuclei (blue), actin filaments (red) and viral particles (green), respectively. Images were taken by CLSM and represented as a maximum z- projection of x–y plane (bottom panels) and x–z plane (upper panels). C–E) The infection is inhibited by EIPA. C) Pretreated cells (20 µM EIPA) were infected (MOI 1) for 16 h and analyzed by immunoblotting with an anti-p72 and an anti-ASFV polyclonal antibodies. D) Pretreated cells (60 µM EIPA) were infected (MOI 5) and stained with Topro3, TRITC-phalloidin and anti-p72. Images were taken by CLSM (mid z-section). Arrowheads: viral factories. E) Supernatants from pretreated (20 µM EIPA) and infected cells (MOI 1) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). F) Bypass of EIPA of ASFV infectivity. Acid mediated bypass was performed and samples of pretreated (20 µM EIPA) and infected cells (MOI 1) for 16 h were analyzed by immunoblotting with an anti-ASFV antibody. G–H) ASFV colocalizes with dextran and induces its uptake. G) Cells were pretreated (60 µM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with 647-dextran for 15 min and analyzed by FACS (n = 3; mean ±S.D.). H) After 30 mpi cells were pulsed with Texas-red-dextran for 15 min and incubated with anti-p72 antibody. Images were taken by CLSM (mid z-section) and Nomarsky. Arrowheads: dextran-virus colocalization. β-actin: load control. S.D., standard deviations. * Unspecific cellular protein detected by the antibody.

Mentions: It has been previously described that after 60 mpi more than 90% of the ASF viral particles are located in the cell [34]. Furthermore, the viral uncoating does not completely occur before 2 hours post infection (hpi) [34]. According to these data, we measured viral uptake by using the specific antibody 17LD3 against p72, the major protein of ASFV capsid [42], [56] (see Materials and Methods and Figure S1A, B and C). Interestingly, amounts of EIPA from 40 µM to 60 µM caused a significant reduction (60%) in the uptake of ASFV infective particles after 60 mpi (Figure 3A), suggesting that ASFV entry depends on Na+/H+ exchanger activity/function.


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)

EIPA treatment inhibits ASFV entry and infection in Vero cells.A–B) EIPA inhibits ASFV uptake. Cells were pretreated (DMSO or EIPA) and infected (MOI 10) for 60 min. A) Infected cells were analyzed by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal. (n = 7, performed in duplicate; mean±S.D.) B) Cells were incubated with Topro3, TRITC-phalloidin and anti-p72 to stain nuclei (blue), actin filaments (red) and viral particles (green), respectively. Images were taken by CLSM and represented as a maximum z- projection of x–y plane (bottom panels) and x–z plane (upper panels). C–E) The infection is inhibited by EIPA. C) Pretreated cells (20 µM EIPA) were infected (MOI 1) for 16 h and analyzed by immunoblotting with an anti-p72 and an anti-ASFV polyclonal antibodies. D) Pretreated cells (60 µM EIPA) were infected (MOI 5) and stained with Topro3, TRITC-phalloidin and anti-p72. Images were taken by CLSM (mid z-section). Arrowheads: viral factories. E) Supernatants from pretreated (20 µM EIPA) and infected cells (MOI 1) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). F) Bypass of EIPA of ASFV infectivity. Acid mediated bypass was performed and samples of pretreated (20 µM EIPA) and infected cells (MOI 1) for 16 h were analyzed by immunoblotting with an anti-ASFV antibody. G–H) ASFV colocalizes with dextran and induces its uptake. G) Cells were pretreated (60 µM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with 647-dextran for 15 min and analyzed by FACS (n = 3; mean ±S.D.). H) After 30 mpi cells were pulsed with Texas-red-dextran for 15 min and incubated with anti-p72 antibody. Images were taken by CLSM (mid z-section) and Nomarsky. Arrowheads: dextran-virus colocalization. β-actin: load control. S.D., standard deviations. * Unspecific cellular protein detected by the antibody.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002754-g003: EIPA treatment inhibits ASFV entry and infection in Vero cells.A–B) EIPA inhibits ASFV uptake. Cells were pretreated (DMSO or EIPA) and infected (MOI 10) for 60 min. A) Infected cells were analyzed by FACS. Graphic shows percentage of virus entry relative to DMSO control, measured as p72 signal. (n = 7, performed in duplicate; mean±S.D.) B) Cells were incubated with Topro3, TRITC-phalloidin and anti-p72 to stain nuclei (blue), actin filaments (red) and viral particles (green), respectively. Images were taken by CLSM and represented as a maximum z- projection of x–y plane (bottom panels) and x–z plane (upper panels). C–E) The infection is inhibited by EIPA. C) Pretreated cells (20 µM EIPA) were infected (MOI 1) for 16 h and analyzed by immunoblotting with an anti-p72 and an anti-ASFV polyclonal antibodies. D) Pretreated cells (60 µM EIPA) were infected (MOI 5) and stained with Topro3, TRITC-phalloidin and anti-p72. Images were taken by CLSM (mid z-section). Arrowheads: viral factories. E) Supernatants from pretreated (20 µM EIPA) and infected cells (MOI 1) were recovered and lytic viruses were titrated (n = 3, mean ±S.D). F) Bypass of EIPA of ASFV infectivity. Acid mediated bypass was performed and samples of pretreated (20 µM EIPA) and infected cells (MOI 1) for 16 h were analyzed by immunoblotting with an anti-ASFV antibody. G–H) ASFV colocalizes with dextran and induces its uptake. G) Cells were pretreated (60 µM EIPA) and infected (MOI 10) or stimulated with PMA for 30 min, pulsed with 647-dextran for 15 min and analyzed by FACS (n = 3; mean ±S.D.). H) After 30 mpi cells were pulsed with Texas-red-dextran for 15 min and incubated with anti-p72 antibody. Images were taken by CLSM (mid z-section) and Nomarsky. Arrowheads: dextran-virus colocalization. β-actin: load control. S.D., standard deviations. * Unspecific cellular protein detected by the antibody.
Mentions: It has been previously described that after 60 mpi more than 90% of the ASF viral particles are located in the cell [34]. Furthermore, the viral uncoating does not completely occur before 2 hours post infection (hpi) [34]. According to these data, we measured viral uptake by using the specific antibody 17LD3 against p72, the major protein of ASFV capsid [42], [56] (see Materials and Methods and Figure S1A, B and C). Interestingly, amounts of EIPA from 40 µM to 60 µM caused a significant reduction (60%) in the uptake of ASFV infective particles after 60 mpi (Figure 3A), suggesting that ASFV entry depends on Na+/H+ exchanger activity/function.

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