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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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Related in: MedlinePlus

Budding of GagΔp6–HrsΔN is dependent on the P(S/T)AP-binding activity of Tsg101. Top, Western blot showing relative levels of Gag–HrsΔN VLP release. Middle, Western blot showing cytoplasmic expression levels of Gag–HrsΔN. Bottom, Anti-Tsg101 Western blot showing depletion of endogenous Tsg101 protein with siRNA, and reexpression of exogenous, siRNA-resistant Tsg101-Flag proteins (Tsg101*). 293T cells were cotransfected as described in the Materials and methods with the following exceptions: (1) lane 1 received no RNA; and (2) lane 5 was a mock transfection.
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fig6: Budding of GagΔp6–HrsΔN is dependent on the P(S/T)AP-binding activity of Tsg101. Top, Western blot showing relative levels of Gag–HrsΔN VLP release. Middle, Western blot showing cytoplasmic expression levels of Gag–HrsΔN. Bottom, Anti-Tsg101 Western blot showing depletion of endogenous Tsg101 protein with siRNA, and reexpression of exogenous, siRNA-resistant Tsg101-Flag proteins (Tsg101*). 293T cells were cotransfected as described in the Materials and methods with the following exceptions: (1) lane 1 received no RNA; and (2) lane 5 was a mock transfection.

Mentions: Next, we examined whether the late domain activity of the HrsΔN polypeptide required the presence of Tsg101. Cellular Tsg101 can be efficiently depleted using RNA interference (Garrus et al., 2001; Fig. 6, bottom, compare lane 1 and lane 2). As shown in Fig. 6, release of GagΔp6–HrsΔN VLPs from 293T cells was blocked when Tsg101 was depleted from the producer cells (Fig. 6, top, compare lane 1 and lane 2). VLP release was restored when an exogenous RNA interference–resistant Tsg101–FLAG protein (denoted Tsg101*) was expressed (Fig. 6, compare lane 2 and lane 3). Therefore, this experiment confirmed that the late domain activity of HrsΔN is dependent on Tsg101.


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)

Budding of GagΔp6–HrsΔN is dependent on the P(S/T)AP-binding activity of Tsg101. Top, Western blot showing relative levels of Gag–HrsΔN VLP release. Middle, Western blot showing cytoplasmic expression levels of Gag–HrsΔN. Bottom, Anti-Tsg101 Western blot showing depletion of endogenous Tsg101 protein with siRNA, and reexpression of exogenous, siRNA-resistant Tsg101-Flag proteins (Tsg101*). 293T cells were cotransfected as described in the Materials and methods with the following exceptions: (1) lane 1 received no RNA; and (2) lane 5 was a mock transfection.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Budding of GagΔp6–HrsΔN is dependent on the P(S/T)AP-binding activity of Tsg101. Top, Western blot showing relative levels of Gag–HrsΔN VLP release. Middle, Western blot showing cytoplasmic expression levels of Gag–HrsΔN. Bottom, Anti-Tsg101 Western blot showing depletion of endogenous Tsg101 protein with siRNA, and reexpression of exogenous, siRNA-resistant Tsg101-Flag proteins (Tsg101*). 293T cells were cotransfected as described in the Materials and methods with the following exceptions: (1) lane 1 received no RNA; and (2) lane 5 was a mock transfection.
Mentions: Next, we examined whether the late domain activity of the HrsΔN polypeptide required the presence of Tsg101. Cellular Tsg101 can be efficiently depleted using RNA interference (Garrus et al., 2001; Fig. 6, bottom, compare lane 1 and lane 2). As shown in Fig. 6, release of GagΔp6–HrsΔN VLPs from 293T cells was blocked when Tsg101 was depleted from the producer cells (Fig. 6, top, compare lane 1 and lane 2). VLP release was restored when an exogenous RNA interference–resistant Tsg101–FLAG protein (denoted Tsg101*) was expressed (Fig. 6, compare lane 2 and lane 3). Therefore, this experiment confirmed that the late domain activity of HrsΔN is dependent on Tsg101.

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