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Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim.

Marshall B, Puthalakath H, Caria S, Chugh S, Doerflinger M, Colman PM, Kvansakul M - Cell Death Dis (2015)

Bottom Line: Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart.Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection.As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, La Trobe University, Kingsbury Drive, Melbourne, 3086 Victoria, Australia [2] La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Melbourne, 3086 Victoria, Australia.

ABSTRACT
Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

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VAR F1L inhibits Bax- but not Bak-mediated apoptosis. (a) Viability of wild-type, Bax−/−, Bak−/− and Bax−/−/Bak−/− DKO cells. MEF cells stably overexpressing VAR F1L or vector, treated with 1.5 μM thapsigargin and cultured for 24 h. (b) Viability of 293T cells transiently overexpressing either BimEL or Bid as well as F1L, Bcl-2 or vector control. Error bars are ±S.E.M. with n=3
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fig3: VAR F1L inhibits Bax- but not Bak-mediated apoptosis. (a) Viability of wild-type, Bax−/−, Bak−/− and Bax−/−/Bak−/− DKO cells. MEF cells stably overexpressing VAR F1L or vector, treated with 1.5 μM thapsigargin and cultured for 24 h. (b) Viability of 293T cells transiently overexpressing either BimEL or Bid as well as F1L, Bcl-2 or vector control. Error bars are ±S.E.M. with n=3

Mentions: To identify differences in anti-apoptotic activity between VAR and VV F1L, we retrovirally transfected VAR F1L into wild-type, Bax−/−, Bak−/− and Bax−/−/Bak−/− DKO cells and treated them with thapsigargin to induced apoptosis via ER stress. Unexpectedly, VAR F1L was unable to protect both wild-type and Bax−/− mouse embryonic fibroblasts (MEFs), whereas Bak−/− MEFs were efficiently protected from apoptosis (Figure 3a) in a manner similar to Bcl-2 (Figure 3b), suggesting that VAR F1L exclusively inhibits Bax-mediated apoptosis. Similar results were obtained after serum withdrawal, with VAR F1L maintaining viability of Bak−/− MEFs but not wild-type and Bax−/− MEFs (Supplementary Figure 5). Overall, the cellular assays mirror our peptide-binding data obtained by ITC, where the lower affinity ligand Bak is not inhibited in a cellular context. Considering the peptide-binding data, we next investigated whether VAR F1L is able to protect against Bim- and Bid-induced apoptosis. 293T cells were transfected with either BimEL or Bid. VAR F1L protected against both BimEL (P=0.006) or Bid-mediated apoptosis (P= 0.0027), with comparable levels of protection observed with Bcl-2.


Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim.

Marshall B, Puthalakath H, Caria S, Chugh S, Doerflinger M, Colman PM, Kvansakul M - Cell Death Dis (2015)

VAR F1L inhibits Bax- but not Bak-mediated apoptosis. (a) Viability of wild-type, Bax−/−, Bak−/− and Bax−/−/Bak−/− DKO cells. MEF cells stably overexpressing VAR F1L or vector, treated with 1.5 μM thapsigargin and cultured for 24 h. (b) Viability of 293T cells transiently overexpressing either BimEL or Bid as well as F1L, Bcl-2 or vector control. Error bars are ±S.E.M. with n=3
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385930&req=5

fig3: VAR F1L inhibits Bax- but not Bak-mediated apoptosis. (a) Viability of wild-type, Bax−/−, Bak−/− and Bax−/−/Bak−/− DKO cells. MEF cells stably overexpressing VAR F1L or vector, treated with 1.5 μM thapsigargin and cultured for 24 h. (b) Viability of 293T cells transiently overexpressing either BimEL or Bid as well as F1L, Bcl-2 or vector control. Error bars are ±S.E.M. with n=3
Mentions: To identify differences in anti-apoptotic activity between VAR and VV F1L, we retrovirally transfected VAR F1L into wild-type, Bax−/−, Bak−/− and Bax−/−/Bak−/− DKO cells and treated them with thapsigargin to induced apoptosis via ER stress. Unexpectedly, VAR F1L was unable to protect both wild-type and Bax−/− mouse embryonic fibroblasts (MEFs), whereas Bak−/− MEFs were efficiently protected from apoptosis (Figure 3a) in a manner similar to Bcl-2 (Figure 3b), suggesting that VAR F1L exclusively inhibits Bax-mediated apoptosis. Similar results were obtained after serum withdrawal, with VAR F1L maintaining viability of Bak−/− MEFs but not wild-type and Bax−/− MEFs (Supplementary Figure 5). Overall, the cellular assays mirror our peptide-binding data obtained by ITC, where the lower affinity ligand Bak is not inhibited in a cellular context. Considering the peptide-binding data, we next investigated whether VAR F1L is able to protect against Bim- and Bid-induced apoptosis. 293T cells were transfected with either BimEL or Bid. VAR F1L protected against both BimEL (P=0.006) or Bid-mediated apoptosis (P= 0.0027), with comparable levels of protection observed with Bcl-2.

Bottom Line: Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart.Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection.As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, La Trobe University, Kingsbury Drive, Melbourne, 3086 Victoria, Australia [2] La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Melbourne, 3086 Victoria, Australia.

ABSTRACT
Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

Show MeSH
Related in: MedlinePlus