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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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Absence of counteracting effect of Vpr on DSB inhibition of HIV-1 VLP assembly and release. Sf9 cells were coinfected with two baculoviruses at equal MOI each (5 PFU/cell), one expressing Pr55Gag, the other expressing His-tagged Vpr. Cells were treated with increasing concentrations of DSB in DMSO aliquots for 30 h at 18 h pi, as indicated on top of the panels. Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-His mAb and phosphatase-labelled anti-mouse IgG antibody, followed by anti-Gag rabbit antibody and peroxidase-labelled anti-rabbit IgG antibody. (A), VLP. (B), WCL. Note the occurrence of Vpr dimer (Vprx2; 30 kDa), stained in blue with the phosphatase reaction. (m), prestained molecular mass markers; (kDa), kiloDaltons.
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Figure 2: Absence of counteracting effect of Vpr on DSB inhibition of HIV-1 VLP assembly and release. Sf9 cells were coinfected with two baculoviruses at equal MOI each (5 PFU/cell), one expressing Pr55Gag, the other expressing His-tagged Vpr. Cells were treated with increasing concentrations of DSB in DMSO aliquots for 30 h at 18 h pi, as indicated on top of the panels. Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-His mAb and phosphatase-labelled anti-mouse IgG antibody, followed by anti-Gag rabbit antibody and peroxidase-labelled anti-rabbit IgG antibody. (A), VLP. (B), WCL. Note the occurrence of Vpr dimer (Vprx2; 30 kDa), stained in blue with the phosphatase reaction. (m), prestained molecular mass markers; (kDa), kiloDaltons.

Mentions: Vpr is coencapsidated with Gag via interaction of the N-terminal alpha-helical domain encompassing residues 17–33 in Vpr [41-44] with the LXXLFG motif in the p6 domain of Gag [21,22,45-48]. In Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vpr, the same DSB sensitivity of VLP assembly was observed as in cells solely expressing AcMNPV-Pr55Gag: both Pr55Gag and Vpr protein signals decreased in parallel and in DSB dose-dependent manner in the extracellular medium of DSB-treated cells, although their intracellular content remained unchanged (Fig. 2). This implied that Vpr did not significantly interfere with the inhibitory effect of DSB on Gag assembly.


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)

Absence of counteracting effect of Vpr on DSB inhibition of HIV-1 VLP assembly and release. Sf9 cells were coinfected with two baculoviruses at equal MOI each (5 PFU/cell), one expressing Pr55Gag, the other expressing His-tagged Vpr. Cells were treated with increasing concentrations of DSB in DMSO aliquots for 30 h at 18 h pi, as indicated on top of the panels. Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-His mAb and phosphatase-labelled anti-mouse IgG antibody, followed by anti-Gag rabbit antibody and peroxidase-labelled anti-rabbit IgG antibody. (A), VLP. (B), WCL. Note the occurrence of Vpr dimer (Vprx2; 30 kDa), stained in blue with the phosphatase reaction. (m), prestained molecular mass markers; (kDa), kiloDaltons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Absence of counteracting effect of Vpr on DSB inhibition of HIV-1 VLP assembly and release. Sf9 cells were coinfected with two baculoviruses at equal MOI each (5 PFU/cell), one expressing Pr55Gag, the other expressing His-tagged Vpr. Cells were treated with increasing concentrations of DSB in DMSO aliquots for 30 h at 18 h pi, as indicated on top of the panels. Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-His mAb and phosphatase-labelled anti-mouse IgG antibody, followed by anti-Gag rabbit antibody and peroxidase-labelled anti-rabbit IgG antibody. (A), VLP. (B), WCL. Note the occurrence of Vpr dimer (Vprx2; 30 kDa), stained in blue with the phosphatase reaction. (m), prestained molecular mass markers; (kDa), kiloDaltons.
Mentions: Vpr is coencapsidated with Gag via interaction of the N-terminal alpha-helical domain encompassing residues 17–33 in Vpr [41-44] with the LXXLFG motif in the p6 domain of Gag [21,22,45-48]. In Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vpr, the same DSB sensitivity of VLP assembly was observed as in cells solely expressing AcMNPV-Pr55Gag: both Pr55Gag and Vpr protein signals decreased in parallel and in DSB dose-dependent manner in the extracellular medium of DSB-treated cells, although their intracellular content remained unchanged (Fig. 2). This implied that Vpr did not significantly interfere with the inhibitory effect of DSB on Gag assembly.

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