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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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Related in: MedlinePlus

Impact of Vpu-sufficiency and -deficiency on the dynamic of HIV-1 infection in hu-mice infected with low dose of virus. (A) Kinetics of plasma viral load was measured by determining RNA copy numbers/ml (log10 values) in plasma at different time following infection of hu-mice with low dose of HIV-1-WT or HIV-1-∆Vpu (n = 4 up to 10-wpi and n = 2 after 10-wpi for HIV-1-WT, and n = 4 for HIV-1-∆Vpu at all time points); the horizontal broken line indicates the detection limit of the viral load assay (40 copies/ml). Viral load for mock-infected animals was less than log10 value of 2. (B and C) T cells in spleen of low dose infected hu-mice were stained at 8 to 10-wpi with a combination of antibodies to identify infected and uninfected CD4+ T cells. (B) Comparison of average frequency of p24+ T cells in spleen of hu-mice infected with HIV-1-WT and HIV-1-∆Vpu (data pooled from 2 independent experiments; n ≥ 5). (C) To determine the impact of Vpu on surface BST2 levels, infected (p24+CD3+CD8-) or uninfected (p24-CD3+CD8-) T cells were gated (first column) and BST2 and CD4 expression was analyzed by two-color dot plots (CD4 versus BST2 (second column) as well as by single color histograms (third and fourth columns). Numbers in the first column represent frequency of p24+ T cells, and numbers in the upper and bottom left quadrants of dot plots represent BST2+CD4- and BST2-CD4- frequency in p24+ T cells. (D) Comparison of relative levels of BST2 on uninfected (p24-) and infected (p24+) T cells from spleen of hu-mice infected with the indicated HIV-1 virus (MFI on p24- T cells = 100%; n ≥ 5;). (E) Bar graph for relative CD4 down regulation on p24+ T cells from hu-mice infected with the indicated HIV-1 virus relative to p24- cells (MFI on p24- T cells = 100%). (F) IFN levels were determined at 8-wpi and 16-wpi in plasma of hu-mice infected with WT or ∆Vpu HIV-1 (for 8-wpi n = 4 for both groups and for 16-wpi n = 2 for WT and n = 4 for ∆Vpu). Error bars represent SD; *, p ≤ 0.05; **, p = 0.002; ***, p ≤ 0.0005. N.S.: not significant.
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Figure 2: Impact of Vpu-sufficiency and -deficiency on the dynamic of HIV-1 infection in hu-mice infected with low dose of virus. (A) Kinetics of plasma viral load was measured by determining RNA copy numbers/ml (log10 values) in plasma at different time following infection of hu-mice with low dose of HIV-1-WT or HIV-1-∆Vpu (n = 4 up to 10-wpi and n = 2 after 10-wpi for HIV-1-WT, and n = 4 for HIV-1-∆Vpu at all time points); the horizontal broken line indicates the detection limit of the viral load assay (40 copies/ml). Viral load for mock-infected animals was less than log10 value of 2. (B and C) T cells in spleen of low dose infected hu-mice were stained at 8 to 10-wpi with a combination of antibodies to identify infected and uninfected CD4+ T cells. (B) Comparison of average frequency of p24+ T cells in spleen of hu-mice infected with HIV-1-WT and HIV-1-∆Vpu (data pooled from 2 independent experiments; n ≥ 5). (C) To determine the impact of Vpu on surface BST2 levels, infected (p24+CD3+CD8-) or uninfected (p24-CD3+CD8-) T cells were gated (first column) and BST2 and CD4 expression was analyzed by two-color dot plots (CD4 versus BST2 (second column) as well as by single color histograms (third and fourth columns). Numbers in the first column represent frequency of p24+ T cells, and numbers in the upper and bottom left quadrants of dot plots represent BST2+CD4- and BST2-CD4- frequency in p24+ T cells. (D) Comparison of relative levels of BST2 on uninfected (p24-) and infected (p24+) T cells from spleen of hu-mice infected with the indicated HIV-1 virus (MFI on p24- T cells = 100%; n ≥ 5;). (E) Bar graph for relative CD4 down regulation on p24+ T cells from hu-mice infected with the indicated HIV-1 virus relative to p24- cells (MFI on p24- T cells = 100%). (F) IFN levels were determined at 8-wpi and 16-wpi in plasma of hu-mice infected with WT or ∆Vpu HIV-1 (for 8-wpi n = 4 for both groups and for 16-wpi n = 2 for WT and n = 4 for ∆Vpu). Error bars represent SD; *, p ≤ 0.05; **, p = 0.002; ***, p ≤ 0.0005. N.S.: not significant.

Mentions: To determine the impact of Vpu on viral replication and propagation under in vivo conditions, we initially infected hu-mice with low dose (~5,000 TCID50) of HIV-1-WT or HIV-1-∆Vpu virus. Hu-mice were bled every alternate week for up to 18 weeks post infection (wpi) for estimation of viral load in plasma and frequency of CD4+ T cells in the blood. As shown in Figure 2A, HIV-1-WT-infected hu-mice showed detectable levels of plasma viral load as early as 2-wpi and it increased further at 4-wpi, a level that was maintained up to 18-wpi. In contrast, HIV-1-∆Vpu infected hu-mice showed delayed and reduced plasma viral load kinetics especially at early time points (2–6 wpi) with peak viral load achieved only between 12- and 16-wpi. Thus at 4- and 18-wpi average plasma viral load in HIV-1-WT infected hu-mice was ~150- and ~5-fold more compared to HIV-1-∆Vpu infected animals. Interestingly, the differences in absolute plasma viremia between the two groups of hu-mice became less significant 14-wpi onwards, indicating that over time HIV-1-∆Vpu replication could reach levels similar to those of HIV-1-WT and suggesting that HIV-1 -∆Vpu virus are ultimately able to overcome host cell restrictions. Analysis of peripheral blood T cells showed that the average frequency of p24+ T cells in blood from HIV-1-WT infected hu-mice was higher than that from their HIV-1-ΔVpu infected counterparts especially at early time points (4-8wpi); however, statistical significance could not be achieved due to large variations in frequency of p24+ T cells in individual hu-mice (Additional file 1: Figure S1A). Detection of infected cells by measurement of virus-encoded GFP could not be used as a substitute as it was less sensitive compared to Gag staining. Moreover, a decrease in CD4+ T cell frequency in blood was observed at 12-wpi and later time points in HIV-1-WT infected hu-mice compared to HIV-1-ΔVpu infected hu-mice (Additional file 1: Figure S1B). This fastest rate of CD4+ T cell depletion by HIV-1 WT virus most probably reflects the more rapid infection dynamic of these viruses relative to their ΔVpu counterparts.


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)

Impact of Vpu-sufficiency and -deficiency on the dynamic of HIV-1 infection in hu-mice infected with low dose of virus. (A) Kinetics of plasma viral load was measured by determining RNA copy numbers/ml (log10 values) in plasma at different time following infection of hu-mice with low dose of HIV-1-WT or HIV-1-∆Vpu (n = 4 up to 10-wpi and n = 2 after 10-wpi for HIV-1-WT, and n = 4 for HIV-1-∆Vpu at all time points); the horizontal broken line indicates the detection limit of the viral load assay (40 copies/ml). Viral load for mock-infected animals was less than log10 value of 2. (B and C) T cells in spleen of low dose infected hu-mice were stained at 8 to 10-wpi with a combination of antibodies to identify infected and uninfected CD4+ T cells. (B) Comparison of average frequency of p24+ T cells in spleen of hu-mice infected with HIV-1-WT and HIV-1-∆Vpu (data pooled from 2 independent experiments; n ≥ 5). (C) To determine the impact of Vpu on surface BST2 levels, infected (p24+CD3+CD8-) or uninfected (p24-CD3+CD8-) T cells were gated (first column) and BST2 and CD4 expression was analyzed by two-color dot plots (CD4 versus BST2 (second column) as well as by single color histograms (third and fourth columns). Numbers in the first column represent frequency of p24+ T cells, and numbers in the upper and bottom left quadrants of dot plots represent BST2+CD4- and BST2-CD4- frequency in p24+ T cells. (D) Comparison of relative levels of BST2 on uninfected (p24-) and infected (p24+) T cells from spleen of hu-mice infected with the indicated HIV-1 virus (MFI on p24- T cells = 100%; n ≥ 5;). (E) Bar graph for relative CD4 down regulation on p24+ T cells from hu-mice infected with the indicated HIV-1 virus relative to p24- cells (MFI on p24- T cells = 100%). (F) IFN levels were determined at 8-wpi and 16-wpi in plasma of hu-mice infected with WT or ∆Vpu HIV-1 (for 8-wpi n = 4 for both groups and for 16-wpi n = 2 for WT and n = 4 for ∆Vpu). Error bars represent SD; *, p ≤ 0.05; **, p = 0.002; ***, p ≤ 0.0005. N.S.: not significant.
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Figure 2: Impact of Vpu-sufficiency and -deficiency on the dynamic of HIV-1 infection in hu-mice infected with low dose of virus. (A) Kinetics of plasma viral load was measured by determining RNA copy numbers/ml (log10 values) in plasma at different time following infection of hu-mice with low dose of HIV-1-WT or HIV-1-∆Vpu (n = 4 up to 10-wpi and n = 2 after 10-wpi for HIV-1-WT, and n = 4 for HIV-1-∆Vpu at all time points); the horizontal broken line indicates the detection limit of the viral load assay (40 copies/ml). Viral load for mock-infected animals was less than log10 value of 2. (B and C) T cells in spleen of low dose infected hu-mice were stained at 8 to 10-wpi with a combination of antibodies to identify infected and uninfected CD4+ T cells. (B) Comparison of average frequency of p24+ T cells in spleen of hu-mice infected with HIV-1-WT and HIV-1-∆Vpu (data pooled from 2 independent experiments; n ≥ 5). (C) To determine the impact of Vpu on surface BST2 levels, infected (p24+CD3+CD8-) or uninfected (p24-CD3+CD8-) T cells were gated (first column) and BST2 and CD4 expression was analyzed by two-color dot plots (CD4 versus BST2 (second column) as well as by single color histograms (third and fourth columns). Numbers in the first column represent frequency of p24+ T cells, and numbers in the upper and bottom left quadrants of dot plots represent BST2+CD4- and BST2-CD4- frequency in p24+ T cells. (D) Comparison of relative levels of BST2 on uninfected (p24-) and infected (p24+) T cells from spleen of hu-mice infected with the indicated HIV-1 virus (MFI on p24- T cells = 100%; n ≥ 5;). (E) Bar graph for relative CD4 down regulation on p24+ T cells from hu-mice infected with the indicated HIV-1 virus relative to p24- cells (MFI on p24- T cells = 100%). (F) IFN levels were determined at 8-wpi and 16-wpi in plasma of hu-mice infected with WT or ∆Vpu HIV-1 (for 8-wpi n = 4 for both groups and for 16-wpi n = 2 for WT and n = 4 for ∆Vpu). Error bars represent SD; *, p ≤ 0.05; **, p = 0.002; ***, p ≤ 0.0005. N.S.: not significant.
Mentions: To determine the impact of Vpu on viral replication and propagation under in vivo conditions, we initially infected hu-mice with low dose (~5,000 TCID50) of HIV-1-WT or HIV-1-∆Vpu virus. Hu-mice were bled every alternate week for up to 18 weeks post infection (wpi) for estimation of viral load in plasma and frequency of CD4+ T cells in the blood. As shown in Figure 2A, HIV-1-WT-infected hu-mice showed detectable levels of plasma viral load as early as 2-wpi and it increased further at 4-wpi, a level that was maintained up to 18-wpi. In contrast, HIV-1-∆Vpu infected hu-mice showed delayed and reduced plasma viral load kinetics especially at early time points (2–6 wpi) with peak viral load achieved only between 12- and 16-wpi. Thus at 4- and 18-wpi average plasma viral load in HIV-1-WT infected hu-mice was ~150- and ~5-fold more compared to HIV-1-∆Vpu infected animals. Interestingly, the differences in absolute plasma viremia between the two groups of hu-mice became less significant 14-wpi onwards, indicating that over time HIV-1-∆Vpu replication could reach levels similar to those of HIV-1-WT and suggesting that HIV-1 -∆Vpu virus are ultimately able to overcome host cell restrictions. Analysis of peripheral blood T cells showed that the average frequency of p24+ T cells in blood from HIV-1-WT infected hu-mice was higher than that from their HIV-1-ΔVpu infected counterparts especially at early time points (4-8wpi); however, statistical significance could not be achieved due to large variations in frequency of p24+ T cells in individual hu-mice (Additional file 1: Figure S1A). Detection of infected cells by measurement of virus-encoded GFP could not be used as a substitute as it was less sensitive compared to Gag staining. Moreover, a decrease in CD4+ T cell frequency in blood was observed at 12-wpi and later time points in HIV-1-WT infected hu-mice compared to HIV-1-ΔVpu infected hu-mice (Additional file 1: Figure S1B). This fastest rate of CD4+ T cell depletion by HIV-1 WT virus most probably reflects the more rapid infection dynamic of these viruses relative to their ΔVpu counterparts.

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