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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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Averaged super-resolution intensity distribution of Env at HIV-1 assembly sites.Image-based, averaged assembly site analysis performed on super-imposed high-resolution images of individual Gag.mEosFP clusters (green) from cells transfected with pCHIV/pCHIVmEosFP (A) or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT) (B), respectively (n = 7 assembly sites per condition) and the averaged Env Alexa Fluor 647 signal in the respective area (red). Graphs show the profile of intensity through the center of the averaged, overlaid intensity images: green line, Gag.mEosFP; red line, Env. Scale bars represent 50 nm.
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ppat-1003198-g007: Averaged super-resolution intensity distribution of Env at HIV-1 assembly sites.Image-based, averaged assembly site analysis performed on super-imposed high-resolution images of individual Gag.mEosFP clusters (green) from cells transfected with pCHIV/pCHIVmEosFP (A) or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT) (B), respectively (n = 7 assembly sites per condition) and the averaged Env Alexa Fluor 647 signal in the respective area (red). Graphs show the profile of intensity through the center of the averaged, overlaid intensity images: green line, Gag.mEosFP; red line, Env. Scale bars represent 50 nm.

Mentions: While providing a view on the whole cell, these computational analyses were not suitable to decipher differences in the Env distribution pattern at specific localizations, e.g. HIV-1 budding sites. To obtain quantitative information on the spatial distribution of Env at such sites, it was required to analyze preselected regions. This was performed by aligning and averaging the intensity distribution of Gag.mEosFP and Env at seven HIV-1 assembly sites each from HeLa cells producing either HIVmEosFP or HIVmEosFPEnv(ΔCT). This procedure should enhance features common to the respective assembly sites, while deemphasizing random distributions. The averaged images of assembly sites from cells producing HIV-1 containing Env(wt) or Env(ΔCT), respectively, showed a very similar pattern for the respective Gag assembly sites but a dramatic difference for the Env distribution pattern (Figure 7). The full-length Env protein (Figure 7A) exhibited a doughnut-shaped average pattern, reflected by a bimodal distribution of Env with a local intensity minimum at the position of the peak of the Gag intensity distribution (Figure 7A). In contrast, Env(ΔCT) (Figure 7B) displayed a more random distribution without any significant enrichment at or close to the actual budding site. The averaged intensity profiles recorded for cells producing the Env(ΔCT) virus revealed no distinctive pattern of Env and no enrichment of Env density in the region of the Gag assembly site (Figure 7B), again confirming the qualitative results obtained by visual inspection of the images.


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)

Averaged super-resolution intensity distribution of Env at HIV-1 assembly sites.Image-based, averaged assembly site analysis performed on super-imposed high-resolution images of individual Gag.mEosFP clusters (green) from cells transfected with pCHIV/pCHIVmEosFP (A) or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT) (B), respectively (n = 7 assembly sites per condition) and the averaged Env Alexa Fluor 647 signal in the respective area (red). Graphs show the profile of intensity through the center of the averaged, overlaid intensity images: green line, Gag.mEosFP; red line, Env. Scale bars represent 50 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585150&req=5

ppat-1003198-g007: Averaged super-resolution intensity distribution of Env at HIV-1 assembly sites.Image-based, averaged assembly site analysis performed on super-imposed high-resolution images of individual Gag.mEosFP clusters (green) from cells transfected with pCHIV/pCHIVmEosFP (A) or pCHIV.Env(ΔCT)/pCHIVmEosFPEnv(ΔCT) (B), respectively (n = 7 assembly sites per condition) and the averaged Env Alexa Fluor 647 signal in the respective area (red). Graphs show the profile of intensity through the center of the averaged, overlaid intensity images: green line, Gag.mEosFP; red line, Env. Scale bars represent 50 nm.
Mentions: While providing a view on the whole cell, these computational analyses were not suitable to decipher differences in the Env distribution pattern at specific localizations, e.g. HIV-1 budding sites. To obtain quantitative information on the spatial distribution of Env at such sites, it was required to analyze preselected regions. This was performed by aligning and averaging the intensity distribution of Gag.mEosFP and Env at seven HIV-1 assembly sites each from HeLa cells producing either HIVmEosFP or HIVmEosFPEnv(ΔCT). This procedure should enhance features common to the respective assembly sites, while deemphasizing random distributions. The averaged images of assembly sites from cells producing HIV-1 containing Env(wt) or Env(ΔCT), respectively, showed a very similar pattern for the respective Gag assembly sites but a dramatic difference for the Env distribution pattern (Figure 7). The full-length Env protein (Figure 7A) exhibited a doughnut-shaped average pattern, reflected by a bimodal distribution of Env with a local intensity minimum at the position of the peak of the Gag intensity distribution (Figure 7A). In contrast, Env(ΔCT) (Figure 7B) displayed a more random distribution without any significant enrichment at or close to the actual budding site. The averaged intensity profiles recorded for cells producing the Env(ΔCT) virus revealed no distinctive pattern of Env and no enrichment of Env density in the region of the Gag assembly site (Figure 7B), again confirming the qualitative results obtained by visual inspection of the images.

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