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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.

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Characterization of CCR5-tropic HIV-1-WT and HIV-1-∆Vpu proviral DNA and viruses. HeLa cells were transfected with WT or Vpu-deficient pNL4.3-Ada-GFP proviral DNA. Transfected cells and virus-containing supernatants were analyzed by western blot using the indicated antibodies (A). Relative virus particle release efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). An aliquot of transfected cells was used for analysis of BST2 levels at the surface of GFP-positive (HIV-expressing cells; dashed line) and -negative (non-transfected cells; continuous line) cells by flow cytometry (representative of n = 3). Gray filled histogram represents preimmune staining with normal rabbit serum (C). Primary CD4+ T cells were isolated from healthy donors and activated with PHA and IL-2. Activated T cells were infected with HIV-1-WT or HIV-1-∆Vpu at an MOI of 1. At different time points post infection, an aliquot of culture was collected for flow cytometry analysis of CD4 and BST2 expression at the surface of GFP-positive (infected; dashed line) or -negative (non infected; continuous line) cells. Data is shown for cells collected 3 days post infection (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 = 2, * p ≤ 0.05, N.S.: not significant). (D). Infectious virus production in supernatant was determined by assessing the levels of luciferase activity (in duplicate) following infection of HeLaTZM-bl reporter cells (E). Data are representative of 3 independent experiments. Error bars represent standard deviations (SD).
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Figure 1: Characterization of CCR5-tropic HIV-1-WT and HIV-1-∆Vpu proviral DNA and viruses. HeLa cells were transfected with WT or Vpu-deficient pNL4.3-Ada-GFP proviral DNA. Transfected cells and virus-containing supernatants were analyzed by western blot using the indicated antibodies (A). Relative virus particle release efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). An aliquot of transfected cells was used for analysis of BST2 levels at the surface of GFP-positive (HIV-expressing cells; dashed line) and -negative (non-transfected cells; continuous line) cells by flow cytometry (representative of n = 3). Gray filled histogram represents preimmune staining with normal rabbit serum (C). Primary CD4+ T cells were isolated from healthy donors and activated with PHA and IL-2. Activated T cells were infected with HIV-1-WT or HIV-1-∆Vpu at an MOI of 1. At different time points post infection, an aliquot of culture was collected for flow cytometry analysis of CD4 and BST2 expression at the surface of GFP-positive (infected; dashed line) or -negative (non infected; continuous line) cells. Data is shown for cells collected 3 days post infection (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 = 2, * p ≤ 0.05, N.S.: not significant). (D). Infectious virus production in supernatant was determined by assessing the levels of luciferase activity (in duplicate) following infection of HeLaTZM-bl reporter cells (E). Data are representative of 3 independent experiments. Error bars represent standard deviations (SD).

Mentions: A group of NSG mice reconstituted with human cord-blood-derived CD34+ stem cells (hu-mice) were infected with CCR5-tropic HIV-1 (pNL4.3-Ada-GFP) expressing Vpu (HIV-1-WT) or lacking Vpu (HIV-1-∆Vpu). It is important to note that the Vpu protein encoded by pNL4.3-Ada-GFP originates from the Ada strain and not the prototypical pNL4.3 variants. Transfection of HeLa cells with WT and ∆Vpu proviral DNA revealed that WT HIV-1 down regulated endogenously expressed surface BST2 in a Vpu-dependent manner and that reduction of BST-2 levels at the surface of HIV-1 producing cells correlated with efficient virus particle release (Figure 1A-C). Furthermore, infection of activated primary human CD4+ T cells also showed a requirement of Vpu in BST2 down regulation and efficient production of infectious virus. Lastly, both viruses had the ability to down regulate the CD4 receptor at the cell surface with what appeared a minor but reproducible contribution resulting from Vpu (Figure 1D-E). However, statistical significance could not be achieved.


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 CCR5-tropic HIV-1-WT and HIV-1-∆Vpu proviral DNA and viruses. HeLa cells were transfected with WT or Vpu-deficient pNL4.3-Ada-GFP proviral DNA. Transfected cells and virus-containing supernatants were analyzed by western blot using the indicated antibodies (A). Relative virus particle release efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). An aliquot of transfected cells was used for analysis of BST2 levels at the surface of GFP-positive (HIV-expressing cells; dashed line) and -negative (non-transfected cells; continuous line) cells by flow cytometry (representative of n = 3). Gray filled histogram represents preimmune staining with normal rabbit serum (C). Primary CD4+ T cells were isolated from healthy donors and activated with PHA and IL-2. Activated T cells were infected with HIV-1-WT or HIV-1-∆Vpu at an MOI of 1. At different time points post infection, an aliquot of culture was collected for flow cytometry analysis of CD4 and BST2 expression at the surface of GFP-positive (infected; dashed line) or -negative (non infected; continuous line) cells. Data is shown for cells collected 3 days post infection (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 = 2, * p ≤ 0.05, N.S.: not significant). (D). Infectious virus production in supernatant was determined by assessing the levels of luciferase activity (in duplicate) following infection of HeLaTZM-bl reporter cells (E). Data are representative of 3 independent experiments. Error bars represent standard deviations (SD).
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Figure 1: Characterization of CCR5-tropic HIV-1-WT and HIV-1-∆Vpu proviral DNA and viruses. HeLa cells were transfected with WT or Vpu-deficient pNL4.3-Ada-GFP proviral DNA. Transfected cells and virus-containing supernatants were analyzed by western blot using the indicated antibodies (A). Relative virus particle release efficiency. The particle release efficiency of HIV-1-WT was set at 100% (average of 3 independent experiments) (B). An aliquot of transfected cells was used for analysis of BST2 levels at the surface of GFP-positive (HIV-expressing cells; dashed line) and -negative (non-transfected cells; continuous line) cells by flow cytometry (representative of n = 3). Gray filled histogram represents preimmune staining with normal rabbit serum (C). Primary CD4+ T cells were isolated from healthy donors and activated with PHA and IL-2. Activated T cells were infected with HIV-1-WT or HIV-1-∆Vpu at an MOI of 1. At different time points post infection, an aliquot of culture was collected for flow cytometry analysis of CD4 and BST2 expression at the surface of GFP-positive (infected; dashed line) or -negative (non infected; continuous line) cells. Data is shown for cells collected 3 days post infection (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 = 2, * p ≤ 0.05, N.S.: not significant). (D). Infectious virus production in supernatant was determined by assessing the levels of luciferase activity (in duplicate) following infection of HeLaTZM-bl reporter cells (E). Data are representative of 3 independent experiments. Error bars represent standard deviations (SD).
Mentions: A group of NSG mice reconstituted with human cord-blood-derived CD34+ stem cells (hu-mice) were infected with CCR5-tropic HIV-1 (pNL4.3-Ada-GFP) expressing Vpu (HIV-1-WT) or lacking Vpu (HIV-1-∆Vpu). It is important to note that the Vpu protein encoded by pNL4.3-Ada-GFP originates from the Ada strain and not the prototypical pNL4.3 variants. Transfection of HeLa cells with WT and ∆Vpu proviral DNA revealed that WT HIV-1 down regulated endogenously expressed surface BST2 in a Vpu-dependent manner and that reduction of BST-2 levels at the surface of HIV-1 producing cells correlated with efficient virus particle release (Figure 1A-C). Furthermore, infection of activated primary human CD4+ T cells also showed a requirement of Vpu in BST2 down regulation and efficient production of infectious virus. Lastly, both viruses had the ability to down regulate the CD4 receptor at the cell surface with what appeared a minor but reproducible contribution resulting from Vpu (Figure 1D-E). However, statistical significance could not be achieved.

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