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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 Env(wt) or Env(ΔCT) in the context of an Env-interaction deficient Gag variant.HeLa cells were transfected with proviral constructs carrying both wt Gag and wt Env (A), wt Gag and Env(ΔCT) (B), Gag carrying the MA mutation and wt Env (C), or comprising both mutated MA and Env(ΔCT) (D), respectively. Fixed cells were stained by indirect immunofluorescence using MAb APR342 and goat anti-mouse Alexa Fluor 532 and MAb 2G12 and goat anti-human Alexa Fluor 647, and subjected to dual-color dSTORM. Scale bar corresponds to 1 µm. An overview of respective cells is presented in Figure S7.
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ppat-1003198-g005: Distribution of Env(wt) or Env(ΔCT) in the context of an Env-interaction deficient Gag variant.HeLa cells were transfected with proviral constructs carrying both wt Gag and wt Env (A), wt Gag and Env(ΔCT) (B), Gag carrying the MA mutation and wt Env (C), or comprising both mutated MA and Env(ΔCT) (D), respectively. Fixed cells were stained by indirect immunofluorescence using MAb APR342 and goat anti-mouse Alexa Fluor 532 and MAb 2G12 and goat anti-human Alexa Fluor 647, and subjected to dual-color dSTORM. Scale bar corresponds to 1 µm. An overview of respective cells is presented in Figure S7.

Mentions: The most likely viral protein responsible for the observed specific Env distribution pattern in the full viral context is Gag since several mutations in its MA domain have been shown to affect Env incorporation [5]–[7], [9]. To directly address this issue, we made use of a panel of proviral constructs carrying either the wt sequence or two point mutations (L8S/S9R) within the MA domain of Gag [8], [40] (designated here as MA(mut)) in the context of Env(wt) or Env(ΔCT). These point mutations have been shown to affect Env(wt) particle incorporation and thereby viral infectivity; both defects are alleviated by truncation of the Env CT [8]. Transfected HeLa cells were fixed and immunostained with antibodies against MA and Env followed by dual-color dSTORM analysis (Figure 5, S7). Typical Gag assembly sites were readily detected in all cases and were associated with Env clusters extending beyond the assembly site for the wt construct (Figure 5A, S7A). These Gag associated Env clusters were absent for MA(wt) in the context of Env(ΔCT) (Figure 5B, S7B) consistent with the results observed above. A similar phenotype was observed for Env(wt) in the context of the MA(mut) virus (Figure 5C, S7C), indicating that the reported Env incorporation defect is due to a loss of Env recruitment to the assembly site. The combination of both mutations displayed an intermediate phenotype, with the overall Env distribution pattern (Figure S7D) resembling the pattern observed for the individual MA or Env mutants, but a slightly more pronounced Env-Gag co-localization revealed upon inspection of individual assembly sites (Figure 5D).


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 Env(wt) or Env(ΔCT) in the context of an Env-interaction deficient Gag variant.HeLa cells were transfected with proviral constructs carrying both wt Gag and wt Env (A), wt Gag and Env(ΔCT) (B), Gag carrying the MA mutation and wt Env (C), or comprising both mutated MA and Env(ΔCT) (D), respectively. Fixed cells were stained by indirect immunofluorescence using MAb APR342 and goat anti-mouse Alexa Fluor 532 and MAb 2G12 and goat anti-human Alexa Fluor 647, and subjected to dual-color dSTORM. Scale bar corresponds to 1 µm. An overview of respective cells is presented in Figure S7.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003198-g005: Distribution of Env(wt) or Env(ΔCT) in the context of an Env-interaction deficient Gag variant.HeLa cells were transfected with proviral constructs carrying both wt Gag and wt Env (A), wt Gag and Env(ΔCT) (B), Gag carrying the MA mutation and wt Env (C), or comprising both mutated MA and Env(ΔCT) (D), respectively. Fixed cells were stained by indirect immunofluorescence using MAb APR342 and goat anti-mouse Alexa Fluor 532 and MAb 2G12 and goat anti-human Alexa Fluor 647, and subjected to dual-color dSTORM. Scale bar corresponds to 1 µm. An overview of respective cells is presented in Figure S7.
Mentions: The most likely viral protein responsible for the observed specific Env distribution pattern in the full viral context is Gag since several mutations in its MA domain have been shown to affect Env incorporation [5]–[7], [9]. To directly address this issue, we made use of a panel of proviral constructs carrying either the wt sequence or two point mutations (L8S/S9R) within the MA domain of Gag [8], [40] (designated here as MA(mut)) in the context of Env(wt) or Env(ΔCT). These point mutations have been shown to affect Env(wt) particle incorporation and thereby viral infectivity; both defects are alleviated by truncation of the Env CT [8]. Transfected HeLa cells were fixed and immunostained with antibodies against MA and Env followed by dual-color dSTORM analysis (Figure 5, S7). Typical Gag assembly sites were readily detected in all cases and were associated with Env clusters extending beyond the assembly site for the wt construct (Figure 5A, S7A). These Gag associated Env clusters were absent for MA(wt) in the context of Env(ΔCT) (Figure 5B, S7B) consistent with the results observed above. A similar phenotype was observed for Env(wt) in the context of the MA(mut) virus (Figure 5C, S7C), indicating that the reported Env incorporation defect is due to a loss of Env recruitment to the assembly site. The combination of both mutations displayed an intermediate phenotype, with the overall Env distribution pattern (Figure S7D) resembling the pattern observed for the individual MA or Env mutants, but a slightly more pronounced Env-Gag co-localization revealed upon inspection of individual assembly sites (Figure 5D).

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