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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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Comparative analysis of Env(wt) distribution expressed in the viral context or alone.(A, B) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in Materials and Methods. (A) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (B) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (C, D) Env distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP (C) or with pEnv(wt) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dual-color super-resolution microscopy as described in Materials and Methods. Scale bars represent 1 µm.
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ppat-1003198-g003: Comparative analysis of Env(wt) distribution expressed in the viral context or alone.(A, B) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in Materials and Methods. (A) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (B) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (C, D) Env distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP (C) or with pEnv(wt) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dual-color super-resolution microscopy as described in Materials and Methods. Scale bars represent 1 µm.

Mentions: The experiments described were carried out employing fixed cells stained with complete IgG molecules, which could potentially affect distribution and detection of the molecules of interest. To exclude potential artifacts, all further experiments (except for those with the MA(mut) constructs, see below) were performed using unfixed cells (stained at 16°C to prevent membrane protein internalization) and immunostaining with Fab fragments. The distribution pattern for Gag assembly sites and Env molecules was largely unaltered when HeLa cells expressing HIVmEosFP were analyzed in this way (Figure 3A) compared to the initial protocol (Figure 2C). Tightly packed clusters of Gag.mEosFP with a diameter of ∼130 nm marked HIV-1 assembly sites, which were overlayed by less dense Env clusters that commonly exhibited enrichment in the periphery and surrounding the Gag budding site (Figure 3B). To investigate whether the membrane distribution of HIV-1 Env was determined by the Env protein itself or was altered by the co-expression of other viral proteins, comparative dSTORM microscopy was performed on HeLa cells expressing either HIVmEosFP as before or only HIV-1 Env in the absence of other viral proteins. Super-resolution imaging of Env alone yielded a clearly different pattern compared to Env in the viral context. The large and distinctive round or doughnut shaped Env clusters, which frequently marked HIV-1 assembly sites (Figure 3C), were not seen in the absence of other viral proteins. Instead, much smaller and more dispersed clusters were observed when Env was expressed alone (Figure 3D). These results demonstrate a clear influence of the other HIV-1 proteins, most likely Gag, on the membrane distribution of HIV-1 Env.


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)

Comparative analysis of Env(wt) distribution expressed in the viral context or alone.(A, B) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in Materials and Methods. (A) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (B) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (C, D) Env distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP (C) or with pEnv(wt) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dual-color super-resolution microscopy as described in Materials and Methods. Scale bars represent 1 µm.
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ppat-1003198-g003: Comparative analysis of Env(wt) distribution expressed in the viral context or alone.(A, B) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in Materials and Methods. (A) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (B) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (C, D) Env distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP (C) or with pEnv(wt) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dual-color super-resolution microscopy as described in Materials and Methods. Scale bars represent 1 µm.
Mentions: The experiments described were carried out employing fixed cells stained with complete IgG molecules, which could potentially affect distribution and detection of the molecules of interest. To exclude potential artifacts, all further experiments (except for those with the MA(mut) constructs, see below) were performed using unfixed cells (stained at 16°C to prevent membrane protein internalization) and immunostaining with Fab fragments. The distribution pattern for Gag assembly sites and Env molecules was largely unaltered when HeLa cells expressing HIVmEosFP were analyzed in this way (Figure 3A) compared to the initial protocol (Figure 2C). Tightly packed clusters of Gag.mEosFP with a diameter of ∼130 nm marked HIV-1 assembly sites, which were overlayed by less dense Env clusters that commonly exhibited enrichment in the periphery and surrounding the Gag budding site (Figure 3B). To investigate whether the membrane distribution of HIV-1 Env was determined by the Env protein itself or was altered by the co-expression of other viral proteins, comparative dSTORM microscopy was performed on HeLa cells expressing either HIVmEosFP as before or only HIV-1 Env in the absence of other viral proteins. Super-resolution imaging of Env alone yielded a clearly different pattern compared to Env in the viral context. The large and distinctive round or doughnut shaped Env clusters, which frequently marked HIV-1 assembly sites (Figure 3C), were not seen in the absence of other viral proteins. Instead, much smaller and more dispersed clusters were observed when Env was expressed alone (Figure 3D). These results demonstrate a clear influence of the other HIV-1 proteins, most likely Gag, on the membrane distribution of HIV-1 Env.

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