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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(ΔCT) 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.Env(ΔCT) and pCHIVmEosFP. Env(ΔCT). 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(ΔCT) 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) Comparison of Env(ΔCT) distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV.Env(ΔCT) and pCHIVmEosFPEnv(ΔCT) (C) or with pEnv(ΔCT) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dSTORM as described. Scale bars represent 1 µm.
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ppat-1003198-g004: Comparative analysis of Env(ΔCT) 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.Env(ΔCT) and pCHIVmEosFP. Env(ΔCT). 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(ΔCT) 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) Comparison of Env(ΔCT) distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV.Env(ΔCT) and pCHIVmEosFPEnv(ΔCT) (C) or with pEnv(ΔCT) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dSTORM as described. Scale bars represent 1 µm.

Mentions: To investigate whether the long Env CT and its presumed interaction with the underlying Gag lattice are important for Env distribution on the surface of HIV-1 producing cells, we performed dual-color super-resolution microscopy on HeLa cells expressing HIVmEosFPEnv(ΔCT). Tightly packed clusters of Gag.mEosFP as described above were detected in this case as well and identified bona fide HIV-1 budding sites (Figure 4A). Clusters of Env(ΔCT) appeared much smaller than observed for wild-type (wt) Env, however. In contrast to (wt) Env clusters, Env(ΔCT) clusters were not enriched at HIV-1 budding sites, but appeared to be more randomly distributed (Figure 4A, B). Furthermore, the characteristic Env clusters observed for the wt protein (compare Figure 3C) were not observed in this case. Similar results were obtained in HIVmEosFP expressing A3.01 cells (Figure S6). Comparison of Env distribution patterns in cells expressing either HIVmEosFPEnv(ΔCT) (Figure 4C) or only Env(ΔCT) (Figure 4D) using super-resolution microscopy revealed no apparent difference with similar cluster size and distribution in both cases. Thus, the characteristic Env distribution appeared to depend on the presence of other viral proteins and on the Env CT.


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(ΔCT) 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.Env(ΔCT) and pCHIVmEosFP. Env(ΔCT). 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(ΔCT) 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) Comparison of Env(ΔCT) distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV.Env(ΔCT) and pCHIVmEosFPEnv(ΔCT) (C) or with pEnv(ΔCT) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dSTORM as described. Scale bars represent 1 µm.
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getmorefigures.php?uid=PMC3585150&req=5

ppat-1003198-g004: Comparative analysis of Env(ΔCT) 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.Env(ΔCT) and pCHIVmEosFP. Env(ΔCT). 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(ΔCT) 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) Comparison of Env(ΔCT) distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV.Env(ΔCT) and pCHIVmEosFPEnv(ΔCT) (C) or with pEnv(ΔCT) (D), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dSTORM as described. Scale bars represent 1 µm.
Mentions: To investigate whether the long Env CT and its presumed interaction with the underlying Gag lattice are important for Env distribution on the surface of HIV-1 producing cells, we performed dual-color super-resolution microscopy on HeLa cells expressing HIVmEosFPEnv(ΔCT). Tightly packed clusters of Gag.mEosFP as described above were detected in this case as well and identified bona fide HIV-1 budding sites (Figure 4A). Clusters of Env(ΔCT) appeared much smaller than observed for wild-type (wt) Env, however. In contrast to (wt) Env clusters, Env(ΔCT) clusters were not enriched at HIV-1 budding sites, but appeared to be more randomly distributed (Figure 4A, B). Furthermore, the characteristic Env clusters observed for the wt protein (compare Figure 3C) were not observed in this case. Similar results were obtained in HIVmEosFP expressing A3.01 cells (Figure S6). Comparison of Env distribution patterns in cells expressing either HIVmEosFPEnv(ΔCT) (Figure 4C) or only Env(ΔCT) (Figure 4D) using super-resolution microscopy revealed no apparent difference with similar cluster size and distribution in both cases. Thus, the characteristic Env distribution appeared to depend on the presence of other viral proteins and on the Env CT.

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