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The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB) is counteracted by Vif and requires its Zinc-binding domain.

Dafonseca S, Coric P, Gay B, Hong SS, Bouaziz S, Boulanger P - Virol. J. (2008)

Bottom Line: DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif.The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H(108)C(114)C(133)H(139) coordinates with a Zn atom.In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université de Lyon I-Claude Bernard, Faculté de Médecine Laënnec, Laboratoire de Virologie & Pathologie Humaine, CNRS FRE-3011, 69372 Lyon Cedex 08, France. sandrinedafonseca@hotmail.com

ABSTRACT

Background: DSB, the 3-O-(3',3'dimethylsuccinyl) derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium.

Results: Wild-type Vif (Vifwt) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H(108)C(114)C(133)H(139) coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone.

Conclusion: The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

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EM and immuno-EM analysis of Pr55Gag-expressing Sf9 cells, with or without Vifwt coexpression. Sf9 cells were infected with AcMNPV-Pr55Gag alone or coinfected with another baculovirus expressing Vif (AcMNPV-Vifwt) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. (a), Control cells expressing Pr55Gag alone; (b), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt. Inset (c), Enlargement of an area of the plasma membrane showing exocytosis of VLP. Note the abundance of VLP at the cell surface in (a), compared to the high VLP content of vesicular compartment in (b). (d, e), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt and harvested at 48 h pi were processed for immuno-EM. Cell sections were incubated with anti-Vif rabbit antibody, followed by 5-nm colloidal gold-tagged anti-rabbit IgG antibody. (d), General view of a cell. The plasma membrane (PM) is materialized by a dotted line; the cytoplasmic area shows vesicles (VS) with intraluminal budding of VLP. (e), Enlargement of VLP-containing vesicles. Note the immunogold labelling of VLP, as well as the accumulation of gold grains at the membrane of VLP-containing vesicles.
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Figure 7: EM and immuno-EM analysis of Pr55Gag-expressing Sf9 cells, with or without Vifwt coexpression. Sf9 cells were infected with AcMNPV-Pr55Gag alone or coinfected with another baculovirus expressing Vif (AcMNPV-Vifwt) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. (a), Control cells expressing Pr55Gag alone; (b), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt. Inset (c), Enlargement of an area of the plasma membrane showing exocytosis of VLP. Note the abundance of VLP at the cell surface in (a), compared to the high VLP content of vesicular compartment in (b). (d, e), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt and harvested at 48 h pi were processed for immuno-EM. Cell sections were incubated with anti-Vif rabbit antibody, followed by 5-nm colloidal gold-tagged anti-rabbit IgG antibody. (d), General view of a cell. The plasma membrane (PM) is materialized by a dotted line; the cytoplasmic area shows vesicles (VS) with intraluminal budding of VLP. (e), Enlargement of VLP-containing vesicles. Note the immunogold labelling of VLP, as well as the accumulation of gold grains at the membrane of VLP-containing vesicles.

Mentions: To further investigate on the mechanism of the DSB counteracting effect of Vif, Sf9 cells coexpressing Pr55Gag and Vifwt or ZBD mutants were analyzed by electron microscopy (EM) and immunoelectron microscopy (immuno-EM). Cells were infected with AcMNPV-Pr55Gag and AcMNPV-Vif, untreated or treated with DSB at 10 μg/ml at 18 h pi, harvested at 48 h pi and processed for EM or immuno-EM using anti-Vif antibody. In control Sf9 cells expressing Pr55Gag alone, the vast majority of VLP assembled at and budded from the plasma membrane (Fig. 7a), as shown in previous studies [16,64,65]. The pattern of VLP assembly and budding was drastically different in Gag+Vifwt-coexpressing cells: VLP were found in abundance in cytoplasmic vesicles (Fig. 7b). Coexpression of Vifwt did not decrease the production of VLP by Pr55Gag-expressing Sf9 cells [24,50], and vesicular VLP egressed into the extracellular medium by exocytosis (Fig. 7c). In immuno-EM, gold grains of anti-Vif antibodies were seen in close association with intravesicular VLP, or along the rim of VLP-containing vesicles (Fig. 7d, e), suggesting that Vif and Pr55Gag proteins colocalized in the same vesicular compartment.


The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB) is counteracted by Vif and requires its Zinc-binding domain.

Dafonseca S, Coric P, Gay B, Hong SS, Bouaziz S, Boulanger P - Virol. J. (2008)

EM and immuno-EM analysis of Pr55Gag-expressing Sf9 cells, with or without Vifwt coexpression. Sf9 cells were infected with AcMNPV-Pr55Gag alone or coinfected with another baculovirus expressing Vif (AcMNPV-Vifwt) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. (a), Control cells expressing Pr55Gag alone; (b), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt. Inset (c), Enlargement of an area of the plasma membrane showing exocytosis of VLP. Note the abundance of VLP at the cell surface in (a), compared to the high VLP content of vesicular compartment in (b). (d, e), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt and harvested at 48 h pi were processed for immuno-EM. Cell sections were incubated with anti-Vif rabbit antibody, followed by 5-nm colloidal gold-tagged anti-rabbit IgG antibody. (d), General view of a cell. The plasma membrane (PM) is materialized by a dotted line; the cytoplasmic area shows vesicles (VS) with intraluminal budding of VLP. (e), Enlargement of VLP-containing vesicles. Note the immunogold labelling of VLP, as well as the accumulation of gold grains at the membrane of VLP-containing vesicles.
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Figure 7: EM and immuno-EM analysis of Pr55Gag-expressing Sf9 cells, with or without Vifwt coexpression. Sf9 cells were infected with AcMNPV-Pr55Gag alone or coinfected with another baculovirus expressing Vif (AcMNPV-Vifwt) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. (a), Control cells expressing Pr55Gag alone; (b), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt. Inset (c), Enlargement of an area of the plasma membrane showing exocytosis of VLP. Note the abundance of VLP at the cell surface in (a), compared to the high VLP content of vesicular compartment in (b). (d, e), Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwt and harvested at 48 h pi were processed for immuno-EM. Cell sections were incubated with anti-Vif rabbit antibody, followed by 5-nm colloidal gold-tagged anti-rabbit IgG antibody. (d), General view of a cell. The plasma membrane (PM) is materialized by a dotted line; the cytoplasmic area shows vesicles (VS) with intraluminal budding of VLP. (e), Enlargement of VLP-containing vesicles. Note the immunogold labelling of VLP, as well as the accumulation of gold grains at the membrane of VLP-containing vesicles.
Mentions: To further investigate on the mechanism of the DSB counteracting effect of Vif, Sf9 cells coexpressing Pr55Gag and Vifwt or ZBD mutants were analyzed by electron microscopy (EM) and immunoelectron microscopy (immuno-EM). Cells were infected with AcMNPV-Pr55Gag and AcMNPV-Vif, untreated or treated with DSB at 10 μg/ml at 18 h pi, harvested at 48 h pi and processed for EM or immuno-EM using anti-Vif antibody. In control Sf9 cells expressing Pr55Gag alone, the vast majority of VLP assembled at and budded from the plasma membrane (Fig. 7a), as shown in previous studies [16,64,65]. The pattern of VLP assembly and budding was drastically different in Gag+Vifwt-coexpressing cells: VLP were found in abundance in cytoplasmic vesicles (Fig. 7b). Coexpression of Vifwt did not decrease the production of VLP by Pr55Gag-expressing Sf9 cells [24,50], and vesicular VLP egressed into the extracellular medium by exocytosis (Fig. 7c). In immuno-EM, gold grains of anti-Vif antibodies were seen in close association with intravesicular VLP, or along the rim of VLP-containing vesicles (Fig. 7d, e), suggesting that Vif and Pr55Gag proteins colocalized in the same vesicular compartment.

Bottom Line: DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif.The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H(108)C(114)C(133)H(139) coordinates with a Zn atom.In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université de Lyon I-Claude Bernard, Faculté de Médecine Laënnec, Laboratoire de Virologie & Pathologie Humaine, CNRS FRE-3011, 69372 Lyon Cedex 08, France. sandrinedafonseca@hotmail.com

ABSTRACT

Background: DSB, the 3-O-(3',3'dimethylsuccinyl) derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium.

Results: Wild-type Vif (Vifwt) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H(108)C(114)C(133)H(139) coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone.

Conclusion: The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

Show MeSH
Related in: MedlinePlus