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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 Gag.mEosFP at the plasma membrane of HeLa cells imaged by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. At 24 hpt, cells were fixed and HIV-1 assembly sites were imaged by PALM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution PALM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. The boxed region in (B) corresponds to the region of interest shown as an enlarged high resolution image in (C). Scale bar in (C) corresponds to 1 µm. (D) Four individual HIV-1 assembly sites from the boxed regions shown in (C). Scale bar corresponds to 100 nm.
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ppat-1003198-g001: Distribution of Gag.mEosFP at the plasma membrane of HeLa cells imaged by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. At 24 hpt, cells were fixed and HIV-1 assembly sites were imaged by PALM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution PALM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. The boxed region in (B) corresponds to the region of interest shown as an enlarged high resolution image in (C). Scale bar in (C) corresponds to 1 µm. (D) Four individual HIV-1 assembly sites from the boxed regions shown in (C). Scale bar corresponds to 100 nm.

Mentions: Total internal fluorescence (TIRF) microscopy and TIRF-PALM imaging of Gag.mEosFP at the plasma membrane of transfected HeLa cells was performed for detection of HIV-1 assembly sites. At the conditions used, Gag.mEosFP was detected with a localization accuracy of ∼28 nm in PALM mode (Figure S1). TIRF microscopy revealed punctuate structures of Gag.mEosFP at the plasma membrane as described previously for Gag.eGFP [36] (Figure 1A). These punctae could be clearly resolved into individual assembly sites by super-resolution imaging (PALM; Figure 1B–D). Compact round assemblies with a diameter of ∼130 nm (Figure 1D), closely resembling assembly sites detected by dSTORM or PALM in previous studies [29], [30] were considered to represent HIV-1 budding structures. The PALM resolution achieved here was not sufficient to detect the semi-spherical architecture of individual Gag shells, which we have recently shown at higher resolution (∼18 nm) by dSTORM imaging of Gag.SNAP assembly sites labelled with a bright synthetic fluorophore [31].


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 Gag.mEosFP at the plasma membrane of HeLa cells imaged by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. At 24 hpt, cells were fixed and HIV-1 assembly sites were imaged by PALM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution PALM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. The boxed region in (B) corresponds to the region of interest shown as an enlarged high resolution image in (C). Scale bar in (C) corresponds to 1 µm. (D) Four individual HIV-1 assembly sites from the boxed regions shown in (C). Scale bar corresponds to 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585150&req=5

ppat-1003198-g001: Distribution of Gag.mEosFP at the plasma membrane of HeLa cells imaged by super-resolution TIRF microscopy.HeLa cells were transfected with equimolar amounts of pCHIV and pCHIVmEosFP. At 24 hpt, cells were fixed and HIV-1 assembly sites were imaged by PALM as described in Materials and Methods. Summed intensity TIRF (A) and high resolution PALM (B) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. The boxed region in (B) corresponds to the region of interest shown as an enlarged high resolution image in (C). Scale bar in (C) corresponds to 1 µm. (D) Four individual HIV-1 assembly sites from the boxed regions shown in (C). Scale bar corresponds to 100 nm.
Mentions: Total internal fluorescence (TIRF) microscopy and TIRF-PALM imaging of Gag.mEosFP at the plasma membrane of transfected HeLa cells was performed for detection of HIV-1 assembly sites. At the conditions used, Gag.mEosFP was detected with a localization accuracy of ∼28 nm in PALM mode (Figure S1). TIRF microscopy revealed punctuate structures of Gag.mEosFP at the plasma membrane as described previously for Gag.eGFP [36] (Figure 1A). These punctae could be clearly resolved into individual assembly sites by super-resolution imaging (PALM; Figure 1B–D). Compact round assemblies with a diameter of ∼130 nm (Figure 1D), closely resembling assembly sites detected by dSTORM or PALM in previous studies [29], [30] were considered to represent HIV-1 budding structures. The PALM resolution achieved here was not sufficient to detect the semi-spherical architecture of individual Gag shells, which we have recently shown at higher resolution (∼18 nm) by dSTORM imaging of Gag.SNAP assembly sites labelled with a bright synthetic fluorophore [31].

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