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Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-terminal tail.

Muranyi W, Malkusch S, Müller B, Heilemann M, Kräusslich HG - PLoS Pathog. (2013)

Bottom Line: Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site.These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated.The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.

ABSTRACT
The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.

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Computational cluster analysis of HIV-1 Env membrane distribution.Global cluster analysis of Env distribution at the plasma membrane was performed based on super-resolution images of HeLa cells transfected with pCHIV/pCHIVmEos.FP, pCHIV. Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), pEnv(wt) or pEnv(ΔCT), or of A3.01 T-cells nucleofected with pCHIV/pCHIVmEos.FP or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), respectively. (A) Image-based, morphological cluster analysis of entire cells. (B) Differential distribution of cluster size for Env(wt) on the surface of HeLa cells in the presence and absence of other viral proteins. The curve was derived by subtraction of the normalized distribution of cluster size obtained by image-based cluster analysis for Env(wt) expressed in the HIV-1 context from that obtained upon expression of Env(wt) alone. (C) Coordinate based all-distance distribution analysis by Ripley's H-function. Statistical evaluation of the maximal H-values [nm] was performed as a correlate of the average cluster diameter. (D) Statistical evaluation of the amplitude of the H-function [a.u.] as a measure of the average degree of clustering. Box plots display 5th percentile, 25th percentile, median (straight line), mean (square), 75th percentile and 95th percentile.
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ppat-1003198-g006: Computational cluster analysis of HIV-1 Env membrane distribution.Global cluster analysis of Env distribution at the plasma membrane was performed based on super-resolution images of HeLa cells transfected with pCHIV/pCHIVmEos.FP, pCHIV. Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), pEnv(wt) or pEnv(ΔCT), or of A3.01 T-cells nucleofected with pCHIV/pCHIVmEos.FP or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), respectively. (A) Image-based, morphological cluster analysis of entire cells. (B) Differential distribution of cluster size for Env(wt) on the surface of HeLa cells in the presence and absence of other viral proteins. The curve was derived by subtraction of the normalized distribution of cluster size obtained by image-based cluster analysis for Env(wt) expressed in the HIV-1 context from that obtained upon expression of Env(wt) alone. (C) Coordinate based all-distance distribution analysis by Ripley's H-function. Statistical evaluation of the maximal H-values [nm] was performed as a correlate of the average cluster diameter. (D) Statistical evaluation of the amplitude of the H-function [a.u.] as a measure of the average degree of clustering. Box plots display 5th percentile, 25th percentile, median (straight line), mean (square), 75th percentile and 95th percentile.

Mentions: To describe the different Env distribution patterns at the cell membrane in an objective and quantitative manner, we performed mathematical cluster analysis using two complementary approaches. Data sets were derived from transfected HeLa or A3.01 cells, respectively; at least three cells per condition were included in the analysis. In a first approach, we used an image-based morphological cluster analysis (Figure 6A) to determine the average cluster size of Env in whole cells. The average cluster size of Env(wt) in virus producing cells was significantly larger than observed for Env(wt) expressed alone or for clusters formed in cells expressing Env(ΔCT) (with or without other viral proteins). The distribution of cluster sizes of Env(wt) in HeLa cells was also analyzed by subtracting values obtained for cells expressing only Env from those obtained for HIV-1 producing cells (Figure 6B). Positive values in this analysis indicate an enrichment of the respective cluster size in virus-producing cells. This analysis revealed that smaller clusters with a radius <50 nm are much more prominent in cells expressing only Env, while larger clusters with a radius of 50 to 150 nm predominate in virus-producing cells.


Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-terminal tail.

Muranyi W, Malkusch S, Müller B, Heilemann M, Kräusslich HG - PLoS Pathog. (2013)

Computational cluster analysis of HIV-1 Env membrane distribution.Global cluster analysis of Env distribution at the plasma membrane was performed based on super-resolution images of HeLa cells transfected with pCHIV/pCHIVmEos.FP, pCHIV. Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), pEnv(wt) or pEnv(ΔCT), or of A3.01 T-cells nucleofected with pCHIV/pCHIVmEos.FP or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), respectively. (A) Image-based, morphological cluster analysis of entire cells. (B) Differential distribution of cluster size for Env(wt) on the surface of HeLa cells in the presence and absence of other viral proteins. The curve was derived by subtraction of the normalized distribution of cluster size obtained by image-based cluster analysis for Env(wt) expressed in the HIV-1 context from that obtained upon expression of Env(wt) alone. (C) Coordinate based all-distance distribution analysis by Ripley's H-function. Statistical evaluation of the maximal H-values [nm] was performed as a correlate of the average cluster diameter. (D) Statistical evaluation of the amplitude of the H-function [a.u.] as a measure of the average degree of clustering. Box plots display 5th percentile, 25th percentile, median (straight line), mean (square), 75th percentile and 95th percentile.
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Related In: Results  -  Collection

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ppat-1003198-g006: Computational cluster analysis of HIV-1 Env membrane distribution.Global cluster analysis of Env distribution at the plasma membrane was performed based on super-resolution images of HeLa cells transfected with pCHIV/pCHIVmEos.FP, pCHIV. Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), pEnv(wt) or pEnv(ΔCT), or of A3.01 T-cells nucleofected with pCHIV/pCHIVmEos.FP or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT), respectively. (A) Image-based, morphological cluster analysis of entire cells. (B) Differential distribution of cluster size for Env(wt) on the surface of HeLa cells in the presence and absence of other viral proteins. The curve was derived by subtraction of the normalized distribution of cluster size obtained by image-based cluster analysis for Env(wt) expressed in the HIV-1 context from that obtained upon expression of Env(wt) alone. (C) Coordinate based all-distance distribution analysis by Ripley's H-function. Statistical evaluation of the maximal H-values [nm] was performed as a correlate of the average cluster diameter. (D) Statistical evaluation of the amplitude of the H-function [a.u.] as a measure of the average degree of clustering. Box plots display 5th percentile, 25th percentile, median (straight line), mean (square), 75th percentile and 95th percentile.
Mentions: To describe the different Env distribution patterns at the cell membrane in an objective and quantitative manner, we performed mathematical cluster analysis using two complementary approaches. Data sets were derived from transfected HeLa or A3.01 cells, respectively; at least three cells per condition were included in the analysis. In a first approach, we used an image-based morphological cluster analysis (Figure 6A) to determine the average cluster size of Env in whole cells. The average cluster size of Env(wt) in virus producing cells was significantly larger than observed for Env(wt) expressed alone or for clusters formed in cells expressing Env(ΔCT) (with or without other viral proteins). The distribution of cluster sizes of Env(wt) in HeLa cells was also analyzed by subtracting values obtained for cells expressing only Env from those obtained for HIV-1 producing cells (Figure 6B). Positive values in this analysis indicate an enrichment of the respective cluster size in virus-producing cells. This analysis revealed that smaller clusters with a radius <50 nm are much more prominent in cells expressing only Env, while larger clusters with a radius of 50 to 150 nm predominate in virus-producing cells.

Bottom Line: Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site.These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated.The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.

ABSTRACT
The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.

Show MeSH
Related in: MedlinePlus