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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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Distribution of HIV-1 Env at the plasma membrane of HeLa cells visualized by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Cells were fixed 24 hpt, stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647 and imaged by dSTORM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution dSTORM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. (C) 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. HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP were fixed and stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647. Cells were subjected to dual-color super-resolution microscopy as described in Materials and Methods. Scale bar corresponds to 1 µm. (D) Individual HIV-1 assembly sites from the boxed regions are shown. Scale bar corresponds to 100 nm. (E) Env surface distribution on extracellular immature HIV-1 particles. Particles were purified from the supernatant of 293T cells transfected with pCHIV carrying a point mutation in the HIV-1 protease active site [39]. An eGFP-tagged version of the accessory protein Vpr [63] was included as a marker to localize the position of individual virions in diffraction limited images. Virions were adhered to fibronectin-coated coverslips, fixed and immunostained with MAb 2G12 and goat anti-human Alexa Fluor 647. The panel shows images of four individual particles with the eGFP signal (green) recorded in diffraction limited mode and the Alexa Fluor 647 (red) signal recorded by dSTORM. Scale bars correspond to 100 nm. (F) Comparison of the Env signal intensities on single extracellular immature particles with the Env signal intensities colocalizing with HIV-1 assembly sites defined by the dSTORM signal for Gag.mEosFP. Fluorescence intensities of the Alexa Fluor 647 signals were determined for the super-resolution images of ten individual particles each. The histogram shows the average values and SD of the fluorescence intensity.
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ppat-1003198-g002: Distribution of HIV-1 Env at the plasma membrane of HeLa cells visualized by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Cells were fixed 24 hpt, stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647 and imaged by dSTORM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution dSTORM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. (C) 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. HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP were fixed and stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647. Cells were subjected to dual-color super-resolution microscopy as described in Materials and Methods. Scale bar corresponds to 1 µm. (D) Individual HIV-1 assembly sites from the boxed regions are shown. Scale bar corresponds to 100 nm. (E) Env surface distribution on extracellular immature HIV-1 particles. Particles were purified from the supernatant of 293T cells transfected with pCHIV carrying a point mutation in the HIV-1 protease active site [39]. An eGFP-tagged version of the accessory protein Vpr [63] was included as a marker to localize the position of individual virions in diffraction limited images. Virions were adhered to fibronectin-coated coverslips, fixed and immunostained with MAb 2G12 and goat anti-human Alexa Fluor 647. The panel shows images of four individual particles with the eGFP signal (green) recorded in diffraction limited mode and the Alexa Fluor 647 (red) signal recorded by dSTORM. Scale bars correspond to 100 nm. (F) Comparison of the Env signal intensities on single extracellular immature particles with the Env signal intensities colocalizing with HIV-1 assembly sites defined by the dSTORM signal for Gag.mEosFP. Fluorescence intensities of the Alexa Fluor 647 signals were determined for the super-resolution images of ten individual particles each. The histogram shows the average values and SD of the fluorescence intensity.

Mentions: HeLa cells transfected as above were fixed and subjected to immunostaining for HIV-1 Env, followed by TIRF- and TIRF-dSTORM microscopy. Alexa Fluor 647, coupled to the protein of interest through the primary and secondary antibody, was detected with a localization accuracy of ∼15 nm in dSTORM mode (Figure S2). TIRF microscopy showed a multi-clustered Env distribution (Figure 2A), similar to previously reported results [21]. Env clusters of various sizes were observed by dSTORM imaging (Figure 2B). These clusters appeared larger and less compact than the Gag.mEosFP assemblies detected in the same cells (compare Figure 1). Dual-color super-resolution microscopy was performed on HeLa cells expressing HIVmEosFP to determine the relative localization of Env with respect to viral Gag assemblies. As shown in the representative images in Figures 2C and 2D, Env clusters surrounding Gag assembly sites were often round and sometimes displayed a doughnut-like shape. A similar pattern was observed when another antibody against gp120 (MAb b12, [37]) was used (Figure S3), or when a harsher fixation protocol reported to block membrane protein motility [38] was applied (Figure S4). Env clusters of similar morphology were also observed in regions lacking a detectable Gag.mEosFP signal (Figure 2C), and only ∼50% of Env clusters were associated with obvious HIV-1 budding sites. Env clusters not associated with characteristic Gag assemblies may correspond to early budding sites with a low number of Gag molecules. Furthermore, co-transfection with a wt plasmid encoding unlabeled Gag was performed in our experiments, and some assembly sites may thus contain a low number of Gag.mEosFP molecules. To address this issue, HeLa cells were transfected with pCHIVmEosFP alone and subsequently processed and analyzed as above. Similar Gag assembly sites surrounded by larger Env clusters were observed, but ∼90% of Env clusters were found to be associated with Gag assemblies in this case (Figure S3).


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)

Distribution of HIV-1 Env at the plasma membrane of HeLa cells visualized by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Cells were fixed 24 hpt, stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647 and imaged by dSTORM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution dSTORM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. (C) 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. HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP were fixed and stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647. Cells were subjected to dual-color super-resolution microscopy as described in Materials and Methods. Scale bar corresponds to 1 µm. (D) Individual HIV-1 assembly sites from the boxed regions are shown. Scale bar corresponds to 100 nm. (E) Env surface distribution on extracellular immature HIV-1 particles. Particles were purified from the supernatant of 293T cells transfected with pCHIV carrying a point mutation in the HIV-1 protease active site [39]. An eGFP-tagged version of the accessory protein Vpr [63] was included as a marker to localize the position of individual virions in diffraction limited images. Virions were adhered to fibronectin-coated coverslips, fixed and immunostained with MAb 2G12 and goat anti-human Alexa Fluor 647. The panel shows images of four individual particles with the eGFP signal (green) recorded in diffraction limited mode and the Alexa Fluor 647 (red) signal recorded by dSTORM. Scale bars correspond to 100 nm. (F) Comparison of the Env signal intensities on single extracellular immature particles with the Env signal intensities colocalizing with HIV-1 assembly sites defined by the dSTORM signal for Gag.mEosFP. Fluorescence intensities of the Alexa Fluor 647 signals were determined for the super-resolution images of ten individual particles each. The histogram shows the average values and SD of the fluorescence intensity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585150&req=5

ppat-1003198-g002: Distribution of HIV-1 Env at the plasma membrane of HeLa cells visualized by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. Cells were fixed 24 hpt, stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647 and imaged by dSTORM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution dSTORM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. (C) 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. HeLa cells transfected with equimolar amounts of pCHIV and pCHIVmEosFP were fixed and stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647. Cells were subjected to dual-color super-resolution microscopy as described in Materials and Methods. Scale bar corresponds to 1 µm. (D) Individual HIV-1 assembly sites from the boxed regions are shown. Scale bar corresponds to 100 nm. (E) Env surface distribution on extracellular immature HIV-1 particles. Particles were purified from the supernatant of 293T cells transfected with pCHIV carrying a point mutation in the HIV-1 protease active site [39]. An eGFP-tagged version of the accessory protein Vpr [63] was included as a marker to localize the position of individual virions in diffraction limited images. Virions were adhered to fibronectin-coated coverslips, fixed and immunostained with MAb 2G12 and goat anti-human Alexa Fluor 647. The panel shows images of four individual particles with the eGFP signal (green) recorded in diffraction limited mode and the Alexa Fluor 647 (red) signal recorded by dSTORM. Scale bars correspond to 100 nm. (F) Comparison of the Env signal intensities on single extracellular immature particles with the Env signal intensities colocalizing with HIV-1 assembly sites defined by the dSTORM signal for Gag.mEosFP. Fluorescence intensities of the Alexa Fluor 647 signals were determined for the super-resolution images of ten individual particles each. The histogram shows the average values and SD of the fluorescence intensity.
Mentions: HeLa cells transfected as above were fixed and subjected to immunostaining for HIV-1 Env, followed by TIRF- and TIRF-dSTORM microscopy. Alexa Fluor 647, coupled to the protein of interest through the primary and secondary antibody, was detected with a localization accuracy of ∼15 nm in dSTORM mode (Figure S2). TIRF microscopy showed a multi-clustered Env distribution (Figure 2A), similar to previously reported results [21]. Env clusters of various sizes were observed by dSTORM imaging (Figure 2B). These clusters appeared larger and less compact than the Gag.mEosFP assemblies detected in the same cells (compare Figure 1). Dual-color super-resolution microscopy was performed on HeLa cells expressing HIVmEosFP to determine the relative localization of Env with respect to viral Gag assemblies. As shown in the representative images in Figures 2C and 2D, Env clusters surrounding Gag assembly sites were often round and sometimes displayed a doughnut-like shape. A similar pattern was observed when another antibody against gp120 (MAb b12, [37]) was used (Figure S3), or when a harsher fixation protocol reported to block membrane protein motility [38] was applied (Figure S4). Env clusters of similar morphology were also observed in regions lacking a detectable Gag.mEosFP signal (Figure 2C), and only ∼50% of Env clusters were associated with obvious HIV-1 budding sites. Env clusters not associated with characteristic Gag assemblies may correspond to early budding sites with a low number of Gag molecules. Furthermore, co-transfection with a wt plasmid encoding unlabeled Gag was performed in our experiments, and some assembly sites may thus contain a low number of Gag.mEosFP molecules. To address this issue, HeLa cells were transfected with pCHIVmEosFP alone and subsequently processed and analyzed as above. Similar Gag assembly sites surrounded by larger Env clusters were observed, but ∼90% of Env clusters were found to be associated with Gag assemblies in this case (Figure S3).

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