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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 can substitute for late domain functions of HIV-1 Gag p6. (A) Summary of the Gag and Gag–Hrs fusion constructs and their VLP-budding phenotypes. (B) The HrsΔN polypeptide rescues the budding arrest caused by mutation of the Gag PTAP late domain. Top, Western blot analysis of VLP release by the Gag–GFP and Gag–Hrs proteins. Bottom, Western blot showing cytoplasmic expression of the Gag–GFP and Gag–Hrs fusion proteins. 293T cells were transfected with 0.5 μg plasmid encoding Gag–GFP (lane 1), GagΔPTAP–GFP (lane 2), GagΔPTAP–HrsΔN (lane 3), GagΔPTAP–HrsΔNΔPSAP (lane 4), GagΔPTAP–HrsΔNΔC (lane 5), or empty vector (lane 6). (C) Trans complementation of deficient GagΔp6 budding. Top, Western blot analyzing VLP release. Bottom, Western blot showing cytoplasmic expression of the different Gag constructs. Cells were cotransfected with 1.5 μg plasmid DNA encoding GagΔp6 and 0.5 μg empty vector (lane 1), Gag–GFP (lane 2), GagΔPTAP–GFP (lane 3), GagΔPTAP–HrsΔN (lane 4), GagΔPTAP–HrsΔNΔPSAP (lane 5), or GagΔPTAP–HrsΔNΔC (lane 6).
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fig4: Hrs can substitute for late domain functions of HIV-1 Gag p6. (A) Summary of the Gag and Gag–Hrs fusion constructs and their VLP-budding phenotypes. (B) The HrsΔN polypeptide rescues the budding arrest caused by mutation of the Gag PTAP late domain. Top, Western blot analysis of VLP release by the Gag–GFP and Gag–Hrs proteins. Bottom, Western blot showing cytoplasmic expression of the Gag–GFP and Gag–Hrs fusion proteins. 293T cells were transfected with 0.5 μg plasmid encoding Gag–GFP (lane 1), GagΔPTAP–GFP (lane 2), GagΔPTAP–HrsΔN (lane 3), GagΔPTAP–HrsΔNΔPSAP (lane 4), GagΔPTAP–HrsΔNΔC (lane 5), or empty vector (lane 6). (C) Trans complementation of deficient GagΔp6 budding. Top, Western blot analyzing VLP release. Bottom, Western blot showing cytoplasmic expression of the different Gag constructs. Cells were cotransfected with 1.5 μg plasmid DNA encoding GagΔp6 and 0.5 μg empty vector (lane 1), Gag–GFP (lane 2), GagΔPTAP–GFP (lane 3), GagΔPTAP–HrsΔN (lane 4), GagΔPTAP–HrsΔNΔPSAP (lane 5), or GagΔPTAP–HrsΔNΔC (lane 6).

Mentions: Human embryonic kidney 293T cells were transfected with HIV Gag expression constructs, and analyzed for protein expression and for VLP release 24–28 h later (Fig. 4). VLP release was analyzed by Western blotting of culture supernatants after sucrose cushion pelleting (Fig. 4, B and C; VLP). Intracellular Gag protein expression levels were analyzed in Western blots of cytoplasmic extracts (Fig. 4, B and C; Cell). As expected, the control Gag–GFP fusion protein expressed well and formed VLPs efficiently (Fig. 4 B, lane 1; Hermida-Matsumoto and Resh, 2000; Garrus et al., 2001). VLP release was dependent on the presence of a functional PTAP late domain, as mutation of the PTAP motif to LIRL (GagΔPTAP–GFP; Fig. 4 B, lane 2) severely attenuated particle release (Göttlinger et al., 1991; Huang et al., 1995; Garrus et al., 2001).


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 can substitute for late domain functions of HIV-1 Gag p6. (A) Summary of the Gag and Gag–Hrs fusion constructs and their VLP-budding phenotypes. (B) The HrsΔN polypeptide rescues the budding arrest caused by mutation of the Gag PTAP late domain. Top, Western blot analysis of VLP release by the Gag–GFP and Gag–Hrs proteins. Bottom, Western blot showing cytoplasmic expression of the Gag–GFP and Gag–Hrs fusion proteins. 293T cells were transfected with 0.5 μg plasmid encoding Gag–GFP (lane 1), GagΔPTAP–GFP (lane 2), GagΔPTAP–HrsΔN (lane 3), GagΔPTAP–HrsΔNΔPSAP (lane 4), GagΔPTAP–HrsΔNΔC (lane 5), or empty vector (lane 6). (C) Trans complementation of deficient GagΔp6 budding. Top, Western blot analyzing VLP release. Bottom, Western blot showing cytoplasmic expression of the different Gag constructs. Cells were cotransfected with 1.5 μg plasmid DNA encoding GagΔp6 and 0.5 μg empty vector (lane 1), Gag–GFP (lane 2), GagΔPTAP–GFP (lane 3), GagΔPTAP–HrsΔN (lane 4), GagΔPTAP–HrsΔNΔPSAP (lane 5), or GagΔPTAP–HrsΔNΔC (lane 6).
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fig4: Hrs can substitute for late domain functions of HIV-1 Gag p6. (A) Summary of the Gag and Gag–Hrs fusion constructs and their VLP-budding phenotypes. (B) The HrsΔN polypeptide rescues the budding arrest caused by mutation of the Gag PTAP late domain. Top, Western blot analysis of VLP release by the Gag–GFP and Gag–Hrs proteins. Bottom, Western blot showing cytoplasmic expression of the Gag–GFP and Gag–Hrs fusion proteins. 293T cells were transfected with 0.5 μg plasmid encoding Gag–GFP (lane 1), GagΔPTAP–GFP (lane 2), GagΔPTAP–HrsΔN (lane 3), GagΔPTAP–HrsΔNΔPSAP (lane 4), GagΔPTAP–HrsΔNΔC (lane 5), or empty vector (lane 6). (C) Trans complementation of deficient GagΔp6 budding. Top, Western blot analyzing VLP release. Bottom, Western blot showing cytoplasmic expression of the different Gag constructs. Cells were cotransfected with 1.5 μg plasmid DNA encoding GagΔp6 and 0.5 μg empty vector (lane 1), Gag–GFP (lane 2), GagΔPTAP–GFP (lane 3), GagΔPTAP–HrsΔN (lane 4), GagΔPTAP–HrsΔNΔPSAP (lane 5), or GagΔPTAP–HrsΔNΔC (lane 6).
Mentions: Human embryonic kidney 293T cells were transfected with HIV Gag expression constructs, and analyzed for protein expression and for VLP release 24–28 h later (Fig. 4). VLP release was analyzed by Western blotting of culture supernatants after sucrose cushion pelleting (Fig. 4, B and C; VLP). Intracellular Gag protein expression levels were analyzed in Western blots of cytoplasmic extracts (Fig. 4, B and C; Cell). As expected, the control Gag–GFP fusion protein expressed well and formed VLPs efficiently (Fig. 4 B, lane 1; Hermida-Matsumoto and Resh, 2000; Garrus et al., 2001). VLP release was dependent on the presence of a functional PTAP late domain, as mutation of the PTAP motif to LIRL (GagΔPTAP–GFP; Fig. 4 B, lane 2) severely attenuated particle release (Göttlinger et al., 1991; Huang et al., 1995; Garrus et al., 2001).

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