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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 analysis of Sf9 cells coexpressing Pr55Gag and ZBD mutants of Vif. Sf9 were coinfected with AcMNPV-Pr55Gag and AcMNPV-VifS116V (a) or AcMNPV-VifC133S (b) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. The vast majority of VLP budding at the plasma membrane was reminiscent of Sf9 cells expressing Pr55Gag alone (refer to Fig. 7a), and contrasted with Sf9 cells coexpressing Pr55Gag and Vifwt (refer to Fig. 7b-e).
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Figure 9: EM analysis of Sf9 cells coexpressing Pr55Gag and ZBD mutants of Vif. Sf9 were coinfected with AcMNPV-Pr55Gag and AcMNPV-VifS116V (a) or AcMNPV-VifC133S (b) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. The vast majority of VLP budding at the plasma membrane was reminiscent of Sf9 cells expressing Pr55Gag alone (refer to Fig. 7a), and contrasted with Sf9 cells coexpressing Pr55Gag and Vifwt (refer to Fig. 7b-e).

Mentions: We next examined cells coexpressing Pr55Gag and ZBD mutants of Vif under the EM, and found that, in the presence of Vif116V and VifC133S, the VLP budding pathway was similar to the one observed in Sf9 cells expressing Pr55Gag alone, i.e. a majority of VLP budding at the plasma membrane and rare intravesicular VLP (less than 10%; Fig. 9). The EM pattern of VifS116V and VifC133S mutants was consistent with their phenotype, as both mutants failed to negate the inhibitory effect of DSB on VLP assembly. Taken together, our results suggested that, in the presence of Vifwt, the VLP assembly and budding process was redirected to the vesicular compartment, and that the VLP egress via exocytosis represented a salvage pathway through which HIV-1 VLP escaped the negative effect of DSB.


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 analysis of Sf9 cells coexpressing Pr55Gag and ZBD mutants of Vif. Sf9 were coinfected with AcMNPV-Pr55Gag and AcMNPV-VifS116V (a) or AcMNPV-VifC133S (b) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. The vast majority of VLP budding at the plasma membrane was reminiscent of Sf9 cells expressing Pr55Gag alone (refer to Fig. 7a), and contrasted with Sf9 cells coexpressing Pr55Gag and Vifwt (refer to Fig. 7b-e).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: EM analysis of Sf9 cells coexpressing Pr55Gag and ZBD mutants of Vif. Sf9 were coinfected with AcMNPV-Pr55Gag and AcMNPV-VifS116V (a) or AcMNPV-VifC133S (b) at equal MOI of each (5 PFU/cell), harvested at 48 h pi, and processed for EM analysis. The vast majority of VLP budding at the plasma membrane was reminiscent of Sf9 cells expressing Pr55Gag alone (refer to Fig. 7a), and contrasted with Sf9 cells coexpressing Pr55Gag and Vifwt (refer to Fig. 7b-e).
Mentions: We next examined cells coexpressing Pr55Gag and ZBD mutants of Vif under the EM, and found that, in the presence of Vif116V and VifC133S, the VLP budding pathway was similar to the one observed in Sf9 cells expressing Pr55Gag alone, i.e. a majority of VLP budding at the plasma membrane and rare intravesicular VLP (less than 10%; Fig. 9). The EM pattern of VifS116V and VifC133S mutants was consistent with their phenotype, as both mutants failed to negate the inhibitory effect of DSB on VLP assembly. Taken together, our results suggested that, in the presence of Vifwt, the VLP assembly and budding process was redirected to the vesicular compartment, and that the VLP egress via exocytosis represented a salvage pathway through which HIV-1 VLP escaped the negative effect of DSB.

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