Limits...
Efficient BST2 antagonism by Vpu is critical for early HIV-1 dissemination in humanized mice.

Dave VP, Hajjar F, Dieng MM, Haddad É, Cohen ÉA - Retrovirology (2013)

Bottom Line: Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2.Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation.Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), 110 Pine avenue west, Montreal, QC H2W 1R7, Canada. eric.cohen@ircm.qc.ca.

ABSTRACT

Background: Vpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(β-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions.

Results: Using a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the β-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation.

Conclusions: Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

Show MeSH

Related in: MedlinePlus

Characterization of HIV-1 proviral DNA encoding the Vpu S52/56 mutant. HeLa cells were transfected with HIV-1-WT, HIV-1-∆Vpu or HIV-1-VpuD52/56 proviral DNA (pNL4.3-Ada-GFP backbone). Transfected cells and virus-containing supernatants were analyzed by Western blot using the indicated antibodies (A). Relative viral particle efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). Analysis of cell-surface BST2 expression by flow cytometry (C). Data are representative of 3 independent experiments. PHA and IL-2 activated primary CD4+ T cells were infected with HIV-1-WT, HIV-1-∆Vpu, or HIV-1-VpuD52/56 virus at an MOI of 1. At different time points post infection, cells were collected for flow cytometric analysis of CD4 and BST2 expression (D). Data is shown for cells collected at 3-dpi. Relative BST2 and CD4 levels at 3-dpi on p24- (open bar) and p24+ (filled bar) cells are shown (MFI on p24-negative = 100%; n = 3). Infectious virus production was determined in supernatants as described in Figure 1(E). Data are representative of 3 independent experiments. Error bar represents SD; * p ≤ 0.05, ** p ≤ 0.005, N.S.: not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4226203&req=5

Figure 4: Characterization of HIV-1 proviral DNA encoding the Vpu S52/56 mutant. HeLa cells were transfected with HIV-1-WT, HIV-1-∆Vpu or HIV-1-VpuD52/56 proviral DNA (pNL4.3-Ada-GFP backbone). Transfected cells and virus-containing supernatants were analyzed by Western blot using the indicated antibodies (A). Relative viral particle efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). Analysis of cell-surface BST2 expression by flow cytometry (C). Data are representative of 3 independent experiments. PHA and IL-2 activated primary CD4+ T cells were infected with HIV-1-WT, HIV-1-∆Vpu, or HIV-1-VpuD52/56 virus at an MOI of 1. At different time points post infection, cells were collected for flow cytometric analysis of CD4 and BST2 expression (D). Data is shown for cells collected at 3-dpi. Relative BST2 and CD4 levels at 3-dpi on p24- (open bar) and p24+ (filled bar) cells are shown (MFI on p24-negative = 100%; n = 3). Infectious virus production was determined in supernatants as described in Figure 1(E). Data are representative of 3 independent experiments. Error bar represents SD; * p ≤ 0.05, ** p ≤ 0.005, N.S.: not significant.

Mentions: While cell culture studies have provided important insight into the mechanistic basis of Vpu-mediated BST2 antagonism and HIV-1 release, it is not clear how Vpu mutations that impair β-TrCP recruitment and inhibit Vpu-mediated BST2 degradation affect HIV-1 infection in vivo. To this end, we mutated the two CTD serine residues at positions 52 and 56 for negatively charged aspartic residues (HIV-1-VpuD52/56) in the context of the pNL4.3-Ada-GFP proviral DNA (HIV-1-WT) since we have previously shown that these substitutions prevented the recruitment of β-TrCP and inhibited BST2 degradation [16]. Transfection of HeLa cells with HIV-1-VpuD52/56 proviral DNA or infection of activated primary human CD4+ T cells with HIV-1-VpuD52/56 revealed that the β-TrCP-binding mutant was strongly attenuated in its ability to promote HIV-1 particle release and down regulate surface BST2, yet was able to down regulate CD4 at the cell surface to levels comparable to Vpu-deficient or proficient viruses. Noticeably, in the context of VpuAda, mutation of the β-TrCP-interacting S52/56 motif did not display residual BST2 antagonism in HeLa cells and showed an impairment of virus production, which was comparable to that of Vpu-defective virus in activated CD4+ T cells (Figure 4).


Efficient BST2 antagonism by Vpu is critical for early HIV-1 dissemination in humanized mice.

Dave VP, Hajjar F, Dieng MM, Haddad É, Cohen ÉA - Retrovirology (2013)

Characterization of HIV-1 proviral DNA encoding the Vpu S52/56 mutant. HeLa cells were transfected with HIV-1-WT, HIV-1-∆Vpu or HIV-1-VpuD52/56 proviral DNA (pNL4.3-Ada-GFP backbone). Transfected cells and virus-containing supernatants were analyzed by Western blot using the indicated antibodies (A). Relative viral particle efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). Analysis of cell-surface BST2 expression by flow cytometry (C). Data are representative of 3 independent experiments. PHA and IL-2 activated primary CD4+ T cells were infected with HIV-1-WT, HIV-1-∆Vpu, or HIV-1-VpuD52/56 virus at an MOI of 1. At different time points post infection, cells were collected for flow cytometric analysis of CD4 and BST2 expression (D). Data is shown for cells collected at 3-dpi. Relative BST2 and CD4 levels at 3-dpi on p24- (open bar) and p24+ (filled bar) cells are shown (MFI on p24-negative = 100%; n = 3). Infectious virus production was determined in supernatants as described in Figure 1(E). Data are representative of 3 independent experiments. Error bar represents SD; * p ≤ 0.05, ** p ≤ 0.005, N.S.: not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4226203&req=5

Figure 4: Characterization of HIV-1 proviral DNA encoding the Vpu S52/56 mutant. HeLa cells were transfected with HIV-1-WT, HIV-1-∆Vpu or HIV-1-VpuD52/56 proviral DNA (pNL4.3-Ada-GFP backbone). Transfected cells and virus-containing supernatants were analyzed by Western blot using the indicated antibodies (A). Relative viral particle efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). Analysis of cell-surface BST2 expression by flow cytometry (C). Data are representative of 3 independent experiments. PHA and IL-2 activated primary CD4+ T cells were infected with HIV-1-WT, HIV-1-∆Vpu, or HIV-1-VpuD52/56 virus at an MOI of 1. At different time points post infection, cells were collected for flow cytometric analysis of CD4 and BST2 expression (D). Data is shown for cells collected at 3-dpi. Relative BST2 and CD4 levels at 3-dpi on p24- (open bar) and p24+ (filled bar) cells are shown (MFI on p24-negative = 100%; n = 3). Infectious virus production was determined in supernatants as described in Figure 1(E). Data are representative of 3 independent experiments. Error bar represents SD; * p ≤ 0.05, ** p ≤ 0.005, N.S.: not significant.
Mentions: While cell culture studies have provided important insight into the mechanistic basis of Vpu-mediated BST2 antagonism and HIV-1 release, it is not clear how Vpu mutations that impair β-TrCP recruitment and inhibit Vpu-mediated BST2 degradation affect HIV-1 infection in vivo. To this end, we mutated the two CTD serine residues at positions 52 and 56 for negatively charged aspartic residues (HIV-1-VpuD52/56) in the context of the pNL4.3-Ada-GFP proviral DNA (HIV-1-WT) since we have previously shown that these substitutions prevented the recruitment of β-TrCP and inhibited BST2 degradation [16]. Transfection of HeLa cells with HIV-1-VpuD52/56 proviral DNA or infection of activated primary human CD4+ T cells with HIV-1-VpuD52/56 revealed that the β-TrCP-binding mutant was strongly attenuated in its ability to promote HIV-1 particle release and down regulate surface BST2, yet was able to down regulate CD4 at the cell surface to levels comparable to Vpu-deficient or proficient viruses. Noticeably, in the context of VpuAda, mutation of the β-TrCP-interacting S52/56 motif did not display residual BST2 antagonism in HeLa cells and showed an impairment of virus production, which was comparable to that of Vpu-defective virus in activated CD4+ T cells (Figure 4).

Bottom Line: Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2.Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation.Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), 110 Pine avenue west, Montreal, QC H2W 1R7, Canada. eric.cohen@ircm.qc.ca.

ABSTRACT

Background: Vpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(β-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions.

Results: Using a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the β-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation.

Conclusions: Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

Show MeSH
Related in: MedlinePlus