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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

Identification of two internal sequences within the EnvCT required for retromer binding.A) Antibody-feeding and retrieval assay was performed as described in Figure 4. Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of two independent experiments. Scale bar is 20 microns. B) Cell lysates prepared from cells stably expressing CD8-gp41CT constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Deleting either of two internal regions of the EnvCT abrogates co-IP of Vps26 and Vps35.
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ppat-1004518-g007: Identification of two internal sequences within the EnvCT required for retromer binding.A) Antibody-feeding and retrieval assay was performed as described in Figure 4. Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of two independent experiments. Scale bar is 20 microns. B) Cell lysates prepared from cells stably expressing CD8-gp41CT constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Deleting either of two internal regions of the EnvCT abrogates co-IP of Vps26 and Vps35.

Mentions: It has previously been reported that two adjacent internal regions within the EnvCT may be involved in trafficking Env back to the Golgi complex [39] and influence Env incorporation into virions [40]. To test whether these regions were implicit in retromer-dependent Env sorting, site-directed mutagenesis was used to independently delete these sequences from the CD8-gp41CT protein. The first mutant (termed Δis1) has an internal deletion that removes a 14 amino acid stretch V747-S760 (inclusive of S760). The second mutant (termed Δis2) has an internal deletion that removes 23 amino acids from L761-L783 (inclusive of L783). Both mutants remove amino acids immediately downstream of the CD8-gp41-L753* truncation. Figure 7A and B show that deletion of either is1 or is2 resulted in loss of binding of the EnvCT to retromer and failure to coimmunoprecipitate Vps26 and Vps35. Moreover, both mutants failed to retrieve to the Golgi following endocytosis from the plasma membrane (R values for CD8 and giantin at 120 min: gp41CT  = 0.44+/−0.05; Δis1 = 0.1+/−0.06; Δis2 = 0.08+/−0.07).


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

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

Identification of two internal sequences within the EnvCT required for retromer binding.A) Antibody-feeding and retrieval assay was performed as described in Figure 4. Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of two independent experiments. Scale bar is 20 microns. B) Cell lysates prepared from cells stably expressing CD8-gp41CT constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Deleting either of two internal regions of the EnvCT abrogates co-IP of Vps26 and Vps35.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231165&req=5

ppat-1004518-g007: Identification of two internal sequences within the EnvCT required for retromer binding.A) Antibody-feeding and retrieval assay was performed as described in Figure 4. Panels are maximum intensity projections reconstructed from serial Z sections through the entire volume of the cell. Data are representative of two independent experiments. Scale bar is 20 microns. B) Cell lysates prepared from cells stably expressing CD8-gp41CT constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Deleting either of two internal regions of the EnvCT abrogates co-IP of Vps26 and Vps35.
Mentions: It has previously been reported that two adjacent internal regions within the EnvCT may be involved in trafficking Env back to the Golgi complex [39] and influence Env incorporation into virions [40]. To test whether these regions were implicit in retromer-dependent Env sorting, site-directed mutagenesis was used to independently delete these sequences from the CD8-gp41CT protein. The first mutant (termed Δis1) has an internal deletion that removes a 14 amino acid stretch V747-S760 (inclusive of S760). The second mutant (termed Δis2) has an internal deletion that removes 23 amino acids from L761-L783 (inclusive of L783). Both mutants remove amino acids immediately downstream of the CD8-gp41-L753* truncation. Figure 7A and B show that deletion of either is1 or is2 resulted in loss of binding of the EnvCT to retromer and failure to coimmunoprecipitate Vps26 and Vps35. Moreover, both mutants failed to retrieve to the Golgi following endocytosis from the plasma membrane (R values for CD8 and giantin at 120 min: gp41CT  = 0.44+/−0.05; Δis1 = 0.1+/−0.06; Δis2 = 0.08+/−0.07).

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