Limits...
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.

Show MeSH

Related in: MedlinePlus

The Tsg101 UEV domain binds the P(S/T)AP motifs from HIV-1 Gag, Tsg101, and Hrs. Biosensor binding isotherms showing the concentration-dependent binding of purified recombinant Tsg101 UEV domain to immobilized fusion peptides spanning the P(S/T)AP motifs of HIV-1 Gag, Tsg101, and Hrs, as well as a mutant form of the HIV-1 Gag PTAP motif (negative control). Solid lines show the optimal fits to simple 1:1 binding models used to obtain binding affinities. Note that substitution of S for T at the second position of the P(S/T)AP motif does not significantly affect the Tsg101 binding affinity (not depicted), but that sequences flanking the central P(S/T)AP tetrapeptides can modulate Tsg101 binding affinity significantly.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172688&req=5

fig2: The Tsg101 UEV domain binds the P(S/T)AP motifs from HIV-1 Gag, Tsg101, and Hrs. Biosensor binding isotherms showing the concentration-dependent binding of purified recombinant Tsg101 UEV domain to immobilized fusion peptides spanning the P(S/T)AP motifs of HIV-1 Gag, Tsg101, and Hrs, as well as a mutant form of the HIV-1 Gag PTAP motif (negative control). Solid lines show the optimal fits to simple 1:1 binding models used to obtain binding affinities. Note that substitution of S for T at the second position of the P(S/T)AP motif does not significantly affect the Tsg101 binding affinity (not depicted), but that sequences flanking the central P(S/T)AP tetrapeptides can modulate Tsg101 binding affinity significantly.

Mentions: First, we tested whether the UEV domain of Tsg101 could bind to isolated P(S/T)AP motifs from HIV-1NL4–3 Gag and from the human Tsg101 and Hrs proteins. The Tsg101 UEV binding epitope is small, and the domain binds with the same affinity to a nine-amino acid peptide as to the intact HIV-1 p6 protein (∼25 μM; Pornillos et al., 2002b). Therefore, 10-amino acid peptides spanning the central P(S/T)AP motifs from Gag, Tsg101, and Hrs were tested for Tsg101 UEV domain binding in BIAcore biosensor experiments. As expected, Tsg101 UEV exhibited concentration-dependent binding to the Gag PTAP peptide, and the equilibrium responses fit a simple 1:1 binding model with a dissociation constant (Kd20°C) of 21 ± 1 μM (Fig. 2). This interaction was specific, as the Tsg101 UEV domain did not bind to a control peptide in which the second Pro residue in the PTAP motif was mutated to Leu (Fig. 2).


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)

The Tsg101 UEV domain binds the P(S/T)AP motifs from HIV-1 Gag, Tsg101, and Hrs. Biosensor binding isotherms showing the concentration-dependent binding of purified recombinant Tsg101 UEV domain to immobilized fusion peptides spanning the P(S/T)AP motifs of HIV-1 Gag, Tsg101, and Hrs, as well as a mutant form of the HIV-1 Gag PTAP motif (negative control). Solid lines show the optimal fits to simple 1:1 binding models used to obtain binding affinities. Note that substitution of S for T at the second position of the P(S/T)AP motif does not significantly affect the Tsg101 binding affinity (not depicted), but that sequences flanking the central P(S/T)AP tetrapeptides can modulate Tsg101 binding affinity significantly.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172688&req=5

fig2: The Tsg101 UEV domain binds the P(S/T)AP motifs from HIV-1 Gag, Tsg101, and Hrs. Biosensor binding isotherms showing the concentration-dependent binding of purified recombinant Tsg101 UEV domain to immobilized fusion peptides spanning the P(S/T)AP motifs of HIV-1 Gag, Tsg101, and Hrs, as well as a mutant form of the HIV-1 Gag PTAP motif (negative control). Solid lines show the optimal fits to simple 1:1 binding models used to obtain binding affinities. Note that substitution of S for T at the second position of the P(S/T)AP motif does not significantly affect the Tsg101 binding affinity (not depicted), but that sequences flanking the central P(S/T)AP tetrapeptides can modulate Tsg101 binding affinity significantly.
Mentions: First, we tested whether the UEV domain of Tsg101 could bind to isolated P(S/T)AP motifs from HIV-1NL4–3 Gag and from the human Tsg101 and Hrs proteins. The Tsg101 UEV binding epitope is small, and the domain binds with the same affinity to a nine-amino acid peptide as to the intact HIV-1 p6 protein (∼25 μM; Pornillos et al., 2002b). Therefore, 10-amino acid peptides spanning the central P(S/T)AP motifs from Gag, Tsg101, and Hrs were tested for Tsg101 UEV domain binding in BIAcore biosensor experiments. As expected, Tsg101 UEV exhibited concentration-dependent binding to the Gag PTAP peptide, and the equilibrium responses fit a simple 1:1 binding model with a dissociation constant (Kd20°C) of 21 ± 1 μM (Fig. 2). This interaction was specific, as the Tsg101 UEV domain did not bind to a control peptide in which the second Pro residue in the PTAP motif was mutated to Leu (Fig. 2).

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