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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

The C terminal 100 amino acids of the Env cytoplasmic tail are required to bind retromer.A) Cells expressing reporter constructs containing truncated EnvCT were treated with control or Vps26 siRNA and an antibody feeding assay was performed to follow Golgi retrieval of endocytosed protein as described in Figure 4. Golgi marker giantin (red) and internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. B) Cells from A stained for Vps26 show that full length and truncated mutants are all efficiently endocytosed and colocalize with intracellular Vps26. Scale bar is 20 microns. C) Truncation of the gp41CT abrogates co-IP of Vps26 and Vps35. Cell lysates prepared from untransfected (UT) or cells stably expressing CD8-reporter constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. D) HeLa cells expressing CD8-reporter constructs were treated with cycloheximide for the indicated periods of time and cell lysates subjected to SDS-PAGE and western blotting. One representative of two independent experiments is shown.
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ppat-1004518-g006: The C terminal 100 amino acids of the Env cytoplasmic tail are required to bind retromer.A) Cells expressing reporter constructs containing truncated EnvCT were treated with control or Vps26 siRNA and an antibody feeding assay was performed to follow Golgi retrieval of endocytosed protein as described in Figure 4. Golgi marker giantin (red) and internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. B) Cells from A stained for Vps26 show that full length and truncated mutants are all efficiently endocytosed and colocalize with intracellular Vps26. Scale bar is 20 microns. C) Truncation of the gp41CT abrogates co-IP of Vps26 and Vps35. Cell lysates prepared from untransfected (UT) or cells stably expressing CD8-reporter constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. D) HeLa cells expressing CD8-reporter constructs were treated with cycloheximide for the indicated periods of time and cell lysates subjected to SDS-PAGE and western blotting. One representative of two independent experiments is shown.

Mentions: A) Amino acid sequence of the NL4.3 HIV-1 cytoplasmic tail. YSPL in bold italics highlights the membrane-proximal YxxL endocytosis motif. The two leucines in bold show the site of truncations of the gp41 cytoplasmic tail in the CD8-gp41 constructs used in Figure 6. B) HeLa TZM-bl cells were transfected with control or Vps26 siRNA and infected with Δ144 NL4.3 lacking the C-terminal 144 amino acids of the Env cytoplasmic tail. Viral supernatants were pelleted and cell lysates prepared and subjected to SDS-PAGE and Western blotting for HIV-1 Env and Gag. One representative western blot from three independent experiments is shown. The band intensities from three independent experiments were quantified using ImageJ and the ratio of HIV-1 Env gp120 to Gag p24CA in virus preparations is shown. Error bars show the SEM. C) Flow cytometry analysis of HIV-1 Env plasma membrane expression following Vps26 KD. Cells were either left uninfected (filled grey), untreated and infected (solid grey line), control siRNA treated and infected (broken black line) or Vps26 siRNA treated and infected (solid black line). One representative from three independent experiments performed with HIV-1 Δ144 (top panel) and WT (lower panel) is shown.


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

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

The C terminal 100 amino acids of the Env cytoplasmic tail are required to bind retromer.A) Cells expressing reporter constructs containing truncated EnvCT were treated with control or Vps26 siRNA and an antibody feeding assay was performed to follow Golgi retrieval of endocytosed protein as described in Figure 4. Golgi marker giantin (red) and internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. B) Cells from A stained for Vps26 show that full length and truncated mutants are all efficiently endocytosed and colocalize with intracellular Vps26. Scale bar is 20 microns. C) Truncation of the gp41CT abrogates co-IP of Vps26 and Vps35. Cell lysates prepared from untransfected (UT) or cells stably expressing CD8-reporter constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. D) HeLa cells expressing CD8-reporter constructs were treated with cycloheximide for the indicated periods of time and cell lysates subjected to SDS-PAGE and western blotting. One representative of two independent experiments is shown.
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Related In: Results  -  Collection

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ppat-1004518-g006: The C terminal 100 amino acids of the Env cytoplasmic tail are required to bind retromer.A) Cells expressing reporter constructs containing truncated EnvCT were treated with control or Vps26 siRNA and an antibody feeding assay was performed to follow Golgi retrieval of endocytosed protein as described in Figure 4. Golgi marker giantin (red) and internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. B) Cells from A stained for Vps26 show that full length and truncated mutants are all efficiently endocytosed and colocalize with intracellular Vps26. Scale bar is 20 microns. C) Truncation of the gp41CT abrogates co-IP of Vps26 and Vps35. Cell lysates prepared from untransfected (UT) or cells stably expressing CD8-reporter constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. D) HeLa cells expressing CD8-reporter constructs were treated with cycloheximide for the indicated periods of time and cell lysates subjected to SDS-PAGE and western blotting. One representative of two independent experiments is shown.
Mentions: A) Amino acid sequence of the NL4.3 HIV-1 cytoplasmic tail. YSPL in bold italics highlights the membrane-proximal YxxL endocytosis motif. The two leucines in bold show the site of truncations of the gp41 cytoplasmic tail in the CD8-gp41 constructs used in Figure 6. B) HeLa TZM-bl cells were transfected with control or Vps26 siRNA and infected with Δ144 NL4.3 lacking the C-terminal 144 amino acids of the Env cytoplasmic tail. Viral supernatants were pelleted and cell lysates prepared and subjected to SDS-PAGE and Western blotting for HIV-1 Env and Gag. One representative western blot from three independent experiments is shown. The band intensities from three independent experiments were quantified using ImageJ and the ratio of HIV-1 Env gp120 to Gag p24CA in virus preparations is shown. Error bars show the SEM. C) Flow cytometry analysis of HIV-1 Env plasma membrane expression following Vps26 KD. Cells were either left uninfected (filled grey), untreated and infected (solid grey line), control siRNA treated and infected (broken black line) or Vps26 siRNA treated and infected (solid black line). One representative from three independent experiments performed with HIV-1 Δ144 (top panel) and WT (lower panel) is shown.

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