Limits...
Retromer regulates HIV-1 envelope glycoprotein trafficking and incorporation into virions.

Groppelli E, Len AC, Granger LA, Jolly C - PLoS Pathog. (2014)

Bottom Line: Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport.Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity.Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, University College London, London, United Kingdom.

ABSTRACT
The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence staining of Env in HIV-1 infected cells.A) HeLa TZM-bl cells were infected with HIV-1, fixed, permeabilized and stained for HIV-1 Env (green) and retromer component Vps26 or the Golgi marker giantin or (red). Panels are single xy slices and are representative examples from three independent experiments. Arrows highlight coincident labeling of Env and Vps26. Scale bar is 20 microns. The amount of immunoreactive Env colocalizing with Vps26 or giantin was calculated from at least 20 cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231165&req=5

ppat-1004518-g002: Immunofluorescence staining of Env in HIV-1 infected cells.A) HeLa TZM-bl cells were infected with HIV-1, fixed, permeabilized and stained for HIV-1 Env (green) and retromer component Vps26 or the Golgi marker giantin or (red). Panels are single xy slices and are representative examples from three independent experiments. Arrows highlight coincident labeling of Env and Vps26. Scale bar is 20 microns. The amount of immunoreactive Env colocalizing with Vps26 or giantin was calculated from at least 20 cells.

Mentions: To further investigate retromer-dependent Env sorting, we performed immunofluorescence microscopy to examine the steady-state localization of Env in virus-infected cells. Cells infected with HIV-1 were fixed, permeabilized and stained for Env (mAb 2G12), the Golgi (giantin) and the retromer component Vps26. Intracellular staining revealed that a proportion of the total immunoreactive Env colocalized with the retromer component Vps26 in HIV-1 infected cells (R value for Env/Vps26  = 0.34+/−0.03) (Figure 2A). This was most clearly seen when examining peripheral Env + cytoplasmic foci. As expected, we also observed that in untreated cells the majority of intracellular Env was concentrated in a perinuclear region and colocalized with the Golgi marker giantin (R value for Env/giantin  = 0.52+/−0.02). That we observed a partial colocalization of Env and retromer is indicative of the dynamic nature of Env trafficking through the endosomal network. Vps26 knockdown did not induce any apparent accumulation of Env in early endosomes or Lamp1 + lysosomes (Figure S3), indicating that Env was not sequestered into intracellular compartments, but rather (as the flow cytometry data suggest) that surface expression was increased after Vps26 knockdown. In agreement with the data showing no effect of Vps26 KD on HIV-1 Gag budding or processing (Figure 1), a punctate cytoplasmic pattern of Gag staining was observed with little or no overlap with Vps26 (Figure S4).


Retromer regulates HIV-1 envelope glycoprotein trafficking and incorporation into virions.

Groppelli E, Len AC, Granger LA, Jolly C - PLoS Pathog. (2014)

Immunofluorescence staining of Env in HIV-1 infected cells.A) HeLa TZM-bl cells were infected with HIV-1, fixed, permeabilized and stained for HIV-1 Env (green) and retromer component Vps26 or the Golgi marker giantin or (red). Panels are single xy slices and are representative examples from three independent experiments. Arrows highlight coincident labeling of Env and Vps26. Scale bar is 20 microns. The amount of immunoreactive Env colocalizing with Vps26 or giantin was calculated from at least 20 cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231165&req=5

ppat-1004518-g002: Immunofluorescence staining of Env in HIV-1 infected cells.A) HeLa TZM-bl cells were infected with HIV-1, fixed, permeabilized and stained for HIV-1 Env (green) and retromer component Vps26 or the Golgi marker giantin or (red). Panels are single xy slices and are representative examples from three independent experiments. Arrows highlight coincident labeling of Env and Vps26. Scale bar is 20 microns. The amount of immunoreactive Env colocalizing with Vps26 or giantin was calculated from at least 20 cells.
Mentions: To further investigate retromer-dependent Env sorting, we performed immunofluorescence microscopy to examine the steady-state localization of Env in virus-infected cells. Cells infected with HIV-1 were fixed, permeabilized and stained for Env (mAb 2G12), the Golgi (giantin) and the retromer component Vps26. Intracellular staining revealed that a proportion of the total immunoreactive Env colocalized with the retromer component Vps26 in HIV-1 infected cells (R value for Env/Vps26  = 0.34+/−0.03) (Figure 2A). This was most clearly seen when examining peripheral Env + cytoplasmic foci. As expected, we also observed that in untreated cells the majority of intracellular Env was concentrated in a perinuclear region and colocalized with the Golgi marker giantin (R value for Env/giantin  = 0.52+/−0.02). That we observed a partial colocalization of Env and retromer is indicative of the dynamic nature of Env trafficking through the endosomal network. Vps26 knockdown did not induce any apparent accumulation of Env in early endosomes or Lamp1 + lysosomes (Figure S3), indicating that Env was not sequestered into intracellular compartments, but rather (as the flow cytometry data suggest) that surface expression was increased after Vps26 knockdown. In agreement with the data showing no effect of Vps26 KD on HIV-1 Gag budding or processing (Figure 1), a punctate cytoplasmic pattern of Gag staining was observed with little or no overlap with Vps26 (Figure S4).

Bottom Line: Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport.Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity.Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection and Immunity, University College London, London, United Kingdom.

ABSTRACT
The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.

No MeSH data available.


Related in: MedlinePlus