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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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Electron microscopic analyses of Gag–GFP and Gag–Hrs budding. (A and B) Transmission electron micrographs of thin-sectioned VLPs released from 293T cells transfected with plasmids encoding Gag–GFP (A) and GagΔPTAP–HrsΔN (B). Note that in both cases, the proteins formed enveloped, spherical VLPs that resembled authentic immature virions except that (1) the electron-dense Gag layer beneath the VLP membrane often appeared discontinuous, suggesting that the COOH-terminal GFP and Hrs polypeptides perturbed the underlying Gag lattice; and (2) VLPs formed by GagΔPTAP–HrsΔN exhibited greater size variation and were, on average, somewhat larger than authentic immature virions. (C–E) Transmission electron micrographs of thin-sectioned 293T cells transfected with plasmids expressing GagΔPTAP–GFP (C), GagΔPTAP–HrsΔNΔC (D), and GagΔPTAP–HrsΔNΔPSAP (E). The top images show clusters of particles associated with the cellular surface, and the bottom images show examples of classic “late domain” phenotypes in which the assembled particles remained tethered to the plasma membrane (or to one another). Bars, 100 nm.
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fig5: Electron microscopic analyses of Gag–GFP and Gag–Hrs budding. (A and B) Transmission electron micrographs of thin-sectioned VLPs released from 293T cells transfected with plasmids encoding Gag–GFP (A) and GagΔPTAP–HrsΔN (B). Note that in both cases, the proteins formed enveloped, spherical VLPs that resembled authentic immature virions except that (1) the electron-dense Gag layer beneath the VLP membrane often appeared discontinuous, suggesting that the COOH-terminal GFP and Hrs polypeptides perturbed the underlying Gag lattice; and (2) VLPs formed by GagΔPTAP–HrsΔN exhibited greater size variation and were, on average, somewhat larger than authentic immature virions. (C–E) Transmission electron micrographs of thin-sectioned 293T cells transfected with plasmids expressing GagΔPTAP–GFP (C), GagΔPTAP–HrsΔNΔC (D), and GagΔPTAP–HrsΔNΔPSAP (E). The top images show clusters of particles associated with the cellular surface, and the bottom images show examples of classic “late domain” phenotypes in which the assembled particles remained tethered to the plasma membrane (or to one another). Bars, 100 nm.

Mentions: Transmission EM was used to confirm that the GagΔPTAP–HrsΔN protein was released in the form of VLPs and to examine the phenotypes of the Gag constructs that failed to bud efficiently. In control experiments, Gag–GFP VLPs appeared as enveloped, spherical particles that generally resembled immature HIV virions in both appearance and size (100–200 nm in diameter; Fig. 5 A). However, the GFP polypeptide appeared to create discontinuities in the Gag–GFP layer, in contrast to the evenly distributed ring of Gag density that is normally observed beneath the membranes of immature HIV-1 virions and Gag VLPs.


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)

Electron microscopic analyses of Gag–GFP and Gag–Hrs budding. (A and B) Transmission electron micrographs of thin-sectioned VLPs released from 293T cells transfected with plasmids encoding Gag–GFP (A) and GagΔPTAP–HrsΔN (B). Note that in both cases, the proteins formed enveloped, spherical VLPs that resembled authentic immature virions except that (1) the electron-dense Gag layer beneath the VLP membrane often appeared discontinuous, suggesting that the COOH-terminal GFP and Hrs polypeptides perturbed the underlying Gag lattice; and (2) VLPs formed by GagΔPTAP–HrsΔN exhibited greater size variation and were, on average, somewhat larger than authentic immature virions. (C–E) Transmission electron micrographs of thin-sectioned 293T cells transfected with plasmids expressing GagΔPTAP–GFP (C), GagΔPTAP–HrsΔNΔC (D), and GagΔPTAP–HrsΔNΔPSAP (E). The top images show clusters of particles associated with the cellular surface, and the bottom images show examples of classic “late domain” phenotypes in which the assembled particles remained tethered to the plasma membrane (or to one another). Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Electron microscopic analyses of Gag–GFP and Gag–Hrs budding. (A and B) Transmission electron micrographs of thin-sectioned VLPs released from 293T cells transfected with plasmids encoding Gag–GFP (A) and GagΔPTAP–HrsΔN (B). Note that in both cases, the proteins formed enveloped, spherical VLPs that resembled authentic immature virions except that (1) the electron-dense Gag layer beneath the VLP membrane often appeared discontinuous, suggesting that the COOH-terminal GFP and Hrs polypeptides perturbed the underlying Gag lattice; and (2) VLPs formed by GagΔPTAP–HrsΔN exhibited greater size variation and were, on average, somewhat larger than authentic immature virions. (C–E) Transmission electron micrographs of thin-sectioned 293T cells transfected with plasmids expressing GagΔPTAP–GFP (C), GagΔPTAP–HrsΔNΔC (D), and GagΔPTAP–HrsΔNΔPSAP (E). The top images show clusters of particles associated with the cellular surface, and the bottom images show examples of classic “late domain” phenotypes in which the assembled particles remained tethered to the plasma membrane (or to one another). Bars, 100 nm.
Mentions: Transmission EM was used to confirm that the GagΔPTAP–HrsΔN protein was released in the form of VLPs and to examine the phenotypes of the Gag constructs that failed to bud efficiently. In control experiments, Gag–GFP VLPs appeared as enveloped, spherical particles that generally resembled immature HIV virions in both appearance and size (100–200 nm in diameter; Fig. 5 A). However, the GFP polypeptide appeared to create discontinuities in the Gag–GFP layer, in contrast to the evenly distributed ring of Gag density that is normally observed beneath the membranes of immature HIV-1 virions and Gag VLPs.

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