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HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein.

Pornillos O, Higginson DS, Stray KM, Fisher RD, Garrus JE, Payne M, He GP, Wang HE, Morham SG, Sundquist WI - J. Cell Biol. (2003)

Bottom Line: Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains.These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane.HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah, School of Medicine, Salt Lake City, UT 84132, USA. wes@biochem.utah.edu

ABSTRACT
The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

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Hrs/Tsg101 yeast two-hybrid binding assays. (A) Filter paper colony lift assay for β-galactosidase reporter gene activity. Yeast cells were cotransformed with pairs of plasmids expressing the indicated Gal4 DNA-binding domain (BD) and activation domain (AD) fusion constructs. Cells were grown on synthetic agar media lacking the appropriate amino acids and were analyzed for β-galactosidase activity (blue) as described in Materials and methods. (B) Binding of Hrs to wild-type and mutant (N45A) Tsg101. Binding experiments in B–D show yeast two-hybrid interactions (together with appropriate controls) as measured in semi-quantitative CPRG β-galactosidase activity assays. Bars depict the average absorbance (595 nm) and SDs from three independent measurements. (C) Binding of wild-type and mutant (ΔPSAP) Hrs to wild-type and mutant (M95A) Tsg101. (D) Binding of Tsg101 to Hrs deletion mutants.
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fig3: Hrs/Tsg101 yeast two-hybrid binding assays. (A) Filter paper colony lift assay for β-galactosidase reporter gene activity. Yeast cells were cotransformed with pairs of plasmids expressing the indicated Gal4 DNA-binding domain (BD) and activation domain (AD) fusion constructs. Cells were grown on synthetic agar media lacking the appropriate amino acids and were analyzed for β-galactosidase activity (blue) as described in Materials and methods. (B) Binding of Hrs to wild-type and mutant (N45A) Tsg101. Binding experiments in B–D show yeast two-hybrid interactions (together with appropriate controls) as measured in semi-quantitative CPRG β-galactosidase activity assays. Bars depict the average absorbance (595 nm) and SDs from three independent measurements. (C) Binding of wild-type and mutant (ΔPSAP) Hrs to wild-type and mutant (M95A) Tsg101. (D) Binding of Tsg101 to Hrs deletion mutants.

Mentions: The interaction of full-length Tsg101 and wild-type Hrs proteins was examined in directed yeast two-hybrid assays. Plasmids expressing a Tsg101–Gal4p binding domain fusion (Tsg101-BD) and an Hrs–Gal4p activation domain fusion (Hrs-AD) were cotransfected into the reporter yeast strain J693, resulting in significant levels of β-galactosidase activity (Fig. 3 A, top left). In contrast, control experiments in which one of the two plasmids encoded only the Gal4p BD or AD alone did not produce significant β-galactosidase activity (Fig. 3 A). Similarly, in semi-quantitative liquid culture assays, the binding of full-length Hrs-AD to full-length Tsg101-BD typically stimulated β-galactosidase activity ∼100-fold above background (Fig. 3, B–D). Therefore, we conclude that the full-length Tsg101 and Hrs proteins interact in the yeast two-hybrid assay.


HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein.

Pornillos O, Higginson DS, Stray KM, Fisher RD, Garrus JE, Payne M, He GP, Wang HE, Morham SG, Sundquist WI - J. Cell Biol. (2003)

Hrs/Tsg101 yeast two-hybrid binding assays. (A) Filter paper colony lift assay for β-galactosidase reporter gene activity. Yeast cells were cotransformed with pairs of plasmids expressing the indicated Gal4 DNA-binding domain (BD) and activation domain (AD) fusion constructs. Cells were grown on synthetic agar media lacking the appropriate amino acids and were analyzed for β-galactosidase activity (blue) as described in Materials and methods. (B) Binding of Hrs to wild-type and mutant (N45A) Tsg101. Binding experiments in B–D show yeast two-hybrid interactions (together with appropriate controls) as measured in semi-quantitative CPRG β-galactosidase activity assays. Bars depict the average absorbance (595 nm) and SDs from three independent measurements. (C) Binding of wild-type and mutant (ΔPSAP) Hrs to wild-type and mutant (M95A) Tsg101. (D) Binding of Tsg101 to Hrs deletion mutants.
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fig3: Hrs/Tsg101 yeast two-hybrid binding assays. (A) Filter paper colony lift assay for β-galactosidase reporter gene activity. Yeast cells were cotransformed with pairs of plasmids expressing the indicated Gal4 DNA-binding domain (BD) and activation domain (AD) fusion constructs. Cells were grown on synthetic agar media lacking the appropriate amino acids and were analyzed for β-galactosidase activity (blue) as described in Materials and methods. (B) Binding of Hrs to wild-type and mutant (N45A) Tsg101. Binding experiments in B–D show yeast two-hybrid interactions (together with appropriate controls) as measured in semi-quantitative CPRG β-galactosidase activity assays. Bars depict the average absorbance (595 nm) and SDs from three independent measurements. (C) Binding of wild-type and mutant (ΔPSAP) Hrs to wild-type and mutant (M95A) Tsg101. (D) Binding of Tsg101 to Hrs deletion mutants.
Mentions: The interaction of full-length Tsg101 and wild-type Hrs proteins was examined in directed yeast two-hybrid assays. Plasmids expressing a Tsg101–Gal4p binding domain fusion (Tsg101-BD) and an Hrs–Gal4p activation domain fusion (Hrs-AD) were cotransfected into the reporter yeast strain J693, resulting in significant levels of β-galactosidase activity (Fig. 3 A, top left). In contrast, control experiments in which one of the two plasmids encoded only the Gal4p BD or AD alone did not produce significant β-galactosidase activity (Fig. 3 A). Similarly, in semi-quantitative liquid culture assays, the binding of full-length Hrs-AD to full-length Tsg101-BD typically stimulated β-galactosidase activity ∼100-fold above background (Fig. 3, B–D). Therefore, we conclude that the full-length Tsg101 and Hrs proteins interact in the yeast two-hybrid assay.

Bottom Line: Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains.These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane.HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah, School of Medicine, Salt Lake City, UT 84132, USA. wes@biochem.utah.edu

ABSTRACT
The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

Show MeSH
Related in: MedlinePlus