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

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.

Figure 1

Effects of DSB on HIV-1 VLP production by (A) insect cells and (B) mammalian cells. (A), Sf9 cells infected with AcMNPV-Pr55Gag were treated with increasing concentrations of DSB in DMSO-aliquots for 30h at 18h pi, as indicated on top of the panels. Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP recovered from the culture medium were analyzed by SDS-PAGE and immunoblotting using anti-Gag polyclonal antibody and phosphatase-labelled anti-rabbit IgG antibody. (i), WCL. (*), Asterisk marks posttranslationally modified Gag precursor (ubiquitinated and/or phosphorylated). This Gag species was not included in the quantification of Pr55Gag polyprotein. (ii), Extracellular VLP. (B), 5BD.1 packaging cells were treated with increasing DSB concentrations in DMSO for 30 h, as indicated on top of the panels, and cells and VLP collected separately and analyzed as above. (i), WCL; (ii), VLP. (iii), Same experiment as in (ii), except for the immunoblot analysis, which was performed using ³⁵S-labelled secondary antibody. Shown in (iii) is an autoradiogram of the blot. Molecular markers (m) were electrophoresed on the left side of the gels, and their molecular masses are indicated in kiloDaltons (kDa).

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.

Figure 3

Influence of Vif on the DSB susceptibility of HIV-1 VLP assembly in Sf9 cells. Sf9 cells were coinfected with equal MOI (5 PFU/cell) of two baculoviruses expressing Pr55Gag and Vif, respectively. Cells were treated with increasing concentrations of DSB in DMSO for 30 h at 18 h pi, as indicated on top of panels (a) and (b), and the x-axis of panel (c). Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting. Blots were reacted with anti-Vif primary antibody and secondary phosphatase-labelled antibody, followed by anti-Gag primary antibody and secondary peroxidase-labelled antibody. (a), WCL. (*), Asterisk marks posttranslationally modified Gag precursor (ubiquitinated and/or phosphorylated). This Gag species was not included in the quantification of Pr55Gag polyprotein. (b), VLP. Molecular mass of prestained markers (m) are indicated in kiloDaltons (kDa) on the left side of panels (a) and (b). (c), Quantification of Gag and Vif proteins in WCL (IC-Gag, intracellular Gag; IC-Vif, intracellular Vif) and extracellular VLP, using SDS-PAGE and radio-immunoblotting. Gag and Vif protein contents were quantified by autoradiography of immunoblots reacted with anti-Gag and anti-Vif rabbit primary antibodies and ³⁵S-labelled secondary anti-rabbit IgG antibody. After autoradiography of the blots, bands of Pr55Gag and Vif proteins were excised and their radioactive content determined by liquid scintillation spectrometry. Results were expressed as percentage of control, untreated samples, which was attributed the 100% value. Mean of three separate experiments ± standard deviation.

Figure 4

Genotype and expression of recombinant Vif mutants in Sf9 cells. (A), Sequence alignment of the central and C-terminal domains of HIV-1 Vif proteins, WT and mutants. The zinc binding domain (ZBD) and its three constitutive loops are boxed: loops 1 and 3 are indicated as dark grey boxes, central loop 2 as a lighter grey box. (B), Cellular expression of recombinant Vif proteins, wild-type and mutants, in baculovirus-infected Sf9 cells. Sf9 cells were infected with baculoviruses (MOI 5) expressing different forms of Vif, as indicated on top of the panel, and harvested at 48 h pi. Whole cell lysates were analyzed by SDS-PAGE and immunoblotting, using anti-Vif primary antibody and secondary peroxidase-labelled antibody. The full-length ZBD mutants VifC133S and Vif116V show an aberrant electrophoretic mobility, as they migrate with a higher apparent molecular weight compared to Vif^wt (23 kDa), and a higher sensitivity to proteolysis, as evidenced by the discrete bands of lower molecular weight breakdown products. Note the propensity of the Vif protein of triple mutant VifsubCΔ170 (20 kDa) to dimerize (Vifx2; 40 kDa).

Figure 5

Counteracting effect of packaging-defective mutant Vif subC on the DSB inhibition of HIV-1 VLP assembly. Sf9 cells were coinfected with two baculoviruses at equal MOI of each (5 PFU/cell), one expressing Pr55Gag, the other expressing the double substitution, packaging-defective mutant VifsubC. Cells were treated with increasing concentrations of DSB in DMSO for 30 h at 18 h pi, as indicated on top of panels (a) and (b), and on the x-axis of panel (c). Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-Vif primary antibody and secondary phosphatase-labelled antibody, followed by anti-Gag primary antibody and secondary peroxidase-labelled antibody. (a), WCL; (b), VLP. Note the low level of Vif protein in VLP, consistent with the packaging-defective phenotype of VifsubC [50]. (m), prestained molecular mass markers; (kDa), kiloDaltons. (c), Quantification of Pr55Gag and Vif protein content of VLP, performed by autoradiography of immunoblots with anti-Gag and anti-Vif rabbit antibodies and ³⁵S-labelled secondary anti-rabbit IgG antibody, as described in the legend to Fig. 3 (c). Results were expressed as percentage of control, untreated samples, which was attributed the 100% value. Mean of three separate experiments ± standard deviation.

Figure 6

Absence of anti-DSB effect of zinc-binding domain mutants of Vif. Sf9 cells were coinfected with two baculoviruses at equal MOI of each (5 PFU/cell), one expressing Pr55Gag, the other expressing VifS116V (A and B, (i)) or VifC133S (A and B, (ii)). Cells were treated with increasing concentrations of DSB in DMSO for 30 h at 18 h pi, as indicated on top of panels (i) and (ii), and on the x-axis of panel (C). Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-Vif primary antibody and secondary peroxidase-labelled antibody, followed by anti-Gag primary antibody and phosphatase-labelled secondary antibody. (A), WCL; (B), VLP. (m), prestained molecular mass markers; (kDa), kiloDaltons. (C), Quantification of VLP produced by DSB-treated Sf9 cells coexpressing Pr55Gag and Vif mutants was performed using SDS-PAGE and autoradiography of immunoblots reacted with anti-Gag and ³⁵S-labelled secondary anti-rabbit IgG antibody, as described in the legends to Fig. 3(c) and 5(c). Results were expressed as percentage of control, untreated samples, which was attributed the 100% value. Mean of three separate experiments ± standard deviation.

Figure 7

EM and immuno-EM analysis of Pr55Gag-expressing Sf9 cells, with or without Vif^wt coexpression. Sf9 cells were infected with AcMNPV-Pr55Gag alone or coinfected with another baculovirus expressing Vif (AcMNPV-Vif^wt) 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-Vif^wt. 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-Vif^wt 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.

Figure 8

DSB treatment of Sf9 cells coexpressing Pr55Gag and Vif^wt. Sf9 coinfected with AcMNPV-Pr55Gag and AcMNPV-Vif^wtat 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.

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 Vif^wt (refer to Fig. 7b-e).