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Modest attenuation of HIV-1 Vpu alleles derived from elite controller plasma.

Chen J, Tibroni N, Sauter D, Galaski J, Miura T, Alter G, Mueller B, Haller C, Walker BD, Kirchhoff F, Brumme ZL, Ueno T, Fackler OT - PLoS ONE (2015)

Bottom Line: EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A.Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences.Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, INF 324, Heidelberg, Germany; German Center for Infection Research, Heidelberg University, Heidelberg. Germany.

ABSTRACT
In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1) can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs) achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP) patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection.

No MeSH data available.


Related in: MedlinePlus

CD4 and CD317/tetherin downregulation by patient-derived Vpus.Surface CD4 and CD317/tetherin levels were analyzed by flow cytometry on A3.01cells 48 h post transfection with the indicated Vpu.GFP expression constructs. A) Representative flow cytometry dot plots of gated living cells for CD4-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD4 cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. B) CD4 cell surface levels of A3.01cells expressing patient-derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. C) Comparison of CD4 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.02), bars represent median and interquartile ranges. D) Representative flow cytometry dot plots of gated living cells for CD317/tetherin-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD317/tetherin cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. E) CD317/tetherin cell surface levels of TZM-bl cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. F) Comparison of CD317 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.64), bars represent median and interquartile ranges.
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pone.0120434.g002: CD4 and CD317/tetherin downregulation by patient-derived Vpus.Surface CD4 and CD317/tetherin levels were analyzed by flow cytometry on A3.01cells 48 h post transfection with the indicated Vpu.GFP expression constructs. A) Representative flow cytometry dot plots of gated living cells for CD4-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD4 cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. B) CD4 cell surface levels of A3.01cells expressing patient-derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. C) Comparison of CD4 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.02), bars represent median and interquartile ranges. D) Representative flow cytometry dot plots of gated living cells for CD317/tetherin-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD317/tetherin cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. E) CD317/tetherin cell surface levels of TZM-bl cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. F) Comparison of CD317 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.64), bars represent median and interquartile ranges.

Mentions: To characterize the biological properties of EC and CP Vpus, these proteins were transiently expressed and subjected to quantitative analysis of established Vpu activities. We first assessed the ability of Vpu to reduce cell surface levels of CD4 in A3.01 cells upon transient transfection of the respective Vpu.GFP expression plasmids. GFP expression allowed to identify transfected cells and to calculate the relative CD4 downregulation activity as the ratio in CD4 mean fluorescence intensity (MFI) between GFP negative and positive cells. The extent of cell surface CD4 downregulation by NL4.3 Vpu (Fig. 2A) was arbitrarily set to 100% and the values obtained upon expression of Vpu alleles expressed relative to this. All patient-derived Vpu.GFP variants displayed robust CD4 downregulation activity (Fig. 2B), albeit with variable efficiency. While almost all CP Vpus analyzed were similarly active in CD4 downmodulation as NL4.3 Vpu (14 of 16), Vpus with intermediate CD4 downregulation activity relative to NL4.3Vpu were more frequent in the EC Vpu group (6 of 15). This resulted in a statistically significant difference between CD4 downregulation by EC (median CD4 downregulation activity relative to NL4.3 116.0% [Interquartile Range (IQR) 77.0%-129.0%]) and CP Vpus (median CD4 downregulation activity relative to NL4.3 135.0% [IQR 119.0%-149.8%]) (p< 0.009; Fig. 2B, 2C). Vpus with slightly reduced CD4 downregulation activity are thus more frequent among ECs than CPs when expressed in A3.01 cells.


Modest attenuation of HIV-1 Vpu alleles derived from elite controller plasma.

Chen J, Tibroni N, Sauter D, Galaski J, Miura T, Alter G, Mueller B, Haller C, Walker BD, Kirchhoff F, Brumme ZL, Ueno T, Fackler OT - PLoS ONE (2015)

CD4 and CD317/tetherin downregulation by patient-derived Vpus.Surface CD4 and CD317/tetherin levels were analyzed by flow cytometry on A3.01cells 48 h post transfection with the indicated Vpu.GFP expression constructs. A) Representative flow cytometry dot plots of gated living cells for CD4-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD4 cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. B) CD4 cell surface levels of A3.01cells expressing patient-derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. C) Comparison of CD4 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.02), bars represent median and interquartile ranges. D) Representative flow cytometry dot plots of gated living cells for CD317/tetherin-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD317/tetherin cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. E) CD317/tetherin cell surface levels of TZM-bl cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. F) Comparison of CD317 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.64), bars represent median and interquartile ranges.
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Related In: Results  -  Collection

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pone.0120434.g002: CD4 and CD317/tetherin downregulation by patient-derived Vpus.Surface CD4 and CD317/tetherin levels were analyzed by flow cytometry on A3.01cells 48 h post transfection with the indicated Vpu.GFP expression constructs. A) Representative flow cytometry dot plots of gated living cells for CD4-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD4 cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. B) CD4 cell surface levels of A3.01cells expressing patient-derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. C) Comparison of CD4 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.02), bars represent median and interquartile ranges. D) Representative flow cytometry dot plots of gated living cells for CD317/tetherin-APC (y-axis) vs. GFP (x-axis). The MFI (Y Geo Mean) of untransfected (left gate) and medium to high GFP-expressing (right gate) cells is indicated. CD317/tetherin cell surface levels of transfected cells were normalized to untransfected cells in the same sample by calculating the ratio of the MFIs of left and right gates and expressed relative to that of NL4.3 Vpu that was arbitrarily set to 100%. E) CD317/tetherin cell surface levels of TZM-bl cells expressing patient derived Vpu proteins relative to NL4.3 Vpu. Shown are mean values of triplicate transfections with the indicated standard deviation. The result is representative of three independent experiments. F) Comparison of CD317 cell surface levels in A3.01 cells expressing EC or CP Vpu alleles. Statistical significance was assessed using two-tailed Mann-Whitney U-Test (p = 0.64), bars represent median and interquartile ranges.
Mentions: To characterize the biological properties of EC and CP Vpus, these proteins were transiently expressed and subjected to quantitative analysis of established Vpu activities. We first assessed the ability of Vpu to reduce cell surface levels of CD4 in A3.01 cells upon transient transfection of the respective Vpu.GFP expression plasmids. GFP expression allowed to identify transfected cells and to calculate the relative CD4 downregulation activity as the ratio in CD4 mean fluorescence intensity (MFI) between GFP negative and positive cells. The extent of cell surface CD4 downregulation by NL4.3 Vpu (Fig. 2A) was arbitrarily set to 100% and the values obtained upon expression of Vpu alleles expressed relative to this. All patient-derived Vpu.GFP variants displayed robust CD4 downregulation activity (Fig. 2B), albeit with variable efficiency. While almost all CP Vpus analyzed were similarly active in CD4 downmodulation as NL4.3 Vpu (14 of 16), Vpus with intermediate CD4 downregulation activity relative to NL4.3Vpu were more frequent in the EC Vpu group (6 of 15). This resulted in a statistically significant difference between CD4 downregulation by EC (median CD4 downregulation activity relative to NL4.3 116.0% [Interquartile Range (IQR) 77.0%-129.0%]) and CP Vpus (median CD4 downregulation activity relative to NL4.3 135.0% [IQR 119.0%-149.8%]) (p< 0.009; Fig. 2B, 2C). Vpus with slightly reduced CD4 downregulation activity are thus more frequent among ECs than CPs when expressed in A3.01 cells.

Bottom Line: EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A.Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences.Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, INF 324, Heidelberg, Germany; German Center for Infection Research, Heidelberg University, Heidelberg. Germany.

ABSTRACT
In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1) can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs) achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP) patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection.

No MeSH data available.


Related in: MedlinePlus