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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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DSB treatment of Sf9 cells coexpressing Pr55Gag and Vifwt. Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwtat equal MOI of each (5 PFU/cell) were treated with DSB at 10 μg/ml for 30 h at 18 h pi. Cells were harvested at 48 h pi, and processed for EM. (a), General view of a cell. (b), Enlargement of a submembranal region of the cell showing VLP in the process of exocytosis. Note the abundance of VLP in the vesicular compartment in panels (a) and (b). VLP-containing vesicles reminiscent of MVBs observed in mammalian cells are indicated with arrows.
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Figure 8: DSB treatment of Sf9 cells coexpressing Pr55Gag and Vifwt. Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwtat equal MOI of each (5 PFU/cell) were treated with DSB at 10 μg/ml for 30 h at 18 h pi. Cells were harvested at 48 h pi, and processed for EM. (a), General view of a cell. (b), Enlargement of a submembranal region of the cell showing VLP in the process of exocytosis. Note the abundance of VLP in the vesicular compartment in panels (a) and (b). VLP-containing vesicles reminiscent of MVBs observed in mammalian cells are indicated with arrows.

Mentions: The proportion of VLP following the intravesicular budding and exocytosis pathway compared to the ones using the plasma membrane pathway was estimated under the EM, by counting several hundreds of VLP in subcellular compartments of more than 20 different cells. In control Sf9 cells expressing Pr55Gag alone, less than 5% VLP were found within the vesicular compartment, whereas in Gag+Vifwt-coexpressing cells, the proportion increased to 30 to 50%, viz. a 5- to 10-fold increase. Likewise, in cells coexpressing Pr55Gag and Vifwt and treated with DSB, most VLP used the intravesicular budding and exocytic pathway (Fig. 8). Interestingly, many VLP-containing vesicles showed an electron-dense, heterogenous lumen (Fig. 8), resembling multivesicular bodies (MVBs) observed in mammalian cells. MVBs belong to the late endosomal subcellular compartment, and have been identified as the preferred budding sites for WT HIV-1 particles in primary human macrophages (reviewed in [66]), as well as in human epithelial and T cells for gag mutants altered in the cluster of basic amino acids of the matrix (MAp17) domain [67].


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)

DSB treatment of Sf9 cells coexpressing Pr55Gag and Vifwt. Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwtat equal MOI of each (5 PFU/cell) were treated with DSB at 10 μg/ml for 30 h at 18 h pi. Cells were harvested at 48 h pi, and processed for EM. (a), General view of a cell. (b), Enlargement of a submembranal region of the cell showing VLP in the process of exocytosis. Note the abundance of VLP in the vesicular compartment in panels (a) and (b). VLP-containing vesicles reminiscent of MVBs observed in mammalian cells are indicated with arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2628355&req=5

Figure 8: DSB treatment of Sf9 cells coexpressing Pr55Gag and Vifwt. Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vifwtat equal MOI of each (5 PFU/cell) were treated with DSB at 10 μg/ml for 30 h at 18 h pi. Cells were harvested at 48 h pi, and processed for EM. (a), General view of a cell. (b), Enlargement of a submembranal region of the cell showing VLP in the process of exocytosis. Note the abundance of VLP in the vesicular compartment in panels (a) and (b). VLP-containing vesicles reminiscent of MVBs observed in mammalian cells are indicated with arrows.
Mentions: The proportion of VLP following the intravesicular budding and exocytosis pathway compared to the ones using the plasma membrane pathway was estimated under the EM, by counting several hundreds of VLP in subcellular compartments of more than 20 different cells. In control Sf9 cells expressing Pr55Gag alone, less than 5% VLP were found within the vesicular compartment, whereas in Gag+Vifwt-coexpressing cells, the proportion increased to 30 to 50%, viz. a 5- to 10-fold increase. Likewise, in cells coexpressing Pr55Gag and Vifwt and treated with DSB, most VLP used the intravesicular budding and exocytic pathway (Fig. 8). Interestingly, many VLP-containing vesicles showed an electron-dense, heterogenous lumen (Fig. 8), resembling multivesicular bodies (MVBs) observed in mammalian cells. MVBs belong to the late endosomal subcellular compartment, and have been identified as the preferred budding sites for WT HIV-1 particles in primary human macrophages (reviewed in [66]), as well as in human epithelial and T cells for gag mutants altered in the cluster of basic amino acids of the matrix (MAp17) domain [67].

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