Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Aug 4;162(3):425-34.
doi: 10.1083/jcb.200302138.

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

Owen Pornillos  1 , Daniel S Higginson, Kirsten M Stray, Robert D Fisher, Jennifer E Garrus, Marielle Payne, Gong-Ping He, Hubert E Wang, Scott G Morham, Wesley I Sundquist
Affiliations

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

Owen Pornillos et al. J Cell Biol. .

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Domain organization of the HIV-1 Gag and human Tsg101 and Hrs proteins. The HIV Gag and Hrs proteins are aligned to emphasize their similarities, with the NH2-terminal membrane-binding domains separated from the COOH-terminal protein–protein interaction domains by a vertical dashed line. UEV, ubiquitin E2 variant; PRD, proline-rich domain; COIL, putative coiled-coil; SBOX, “steadiness box/Vps28 binding site” (Feng et al., 2000). Locations of P(S/T)AP and PPEY motifs are also indicated.
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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).
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
Budding of Gag Δp6 –Hrs ΔN 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.
Figure 7.
Figure 7.
Models for Tsg101 recruitment and activation during MVB and HIV budding. (A) Model illustrating sites of Tsg101/Hrs interaction and a possible activation mechanism for Tsg101 (see text for details). Note that our data allow the possibility that other proteins might bridge or contribute to the interaction between the COOH-terminal regions of Tsg101 and Hrs. (B) Model illustrating how HIV Gag mimics the Tsg101-recruiting function of Hrs and redirects Tsg101 and the ESCRT-I complex to the plasma membrane to facilitate viral budding.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Asao, H., Y. Sasaki, T. Arita, N. Tanaka, K. Endo, H. Kasai, T. Takeshita, Y. Endo, T. Fujita, and K. Sugamura. 1997. Hrs is associated with STAM, a signal-transducing adaptor molecule. Its suppressive effect on cytokine-induced cell growth. J. Biol. Chem. 272:32785–32791. - PubMed
    1. Babst, M., D. Katzmann, E. Estepa-Sabal, T. Meerloo, and S. Emr. 2002. a. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell. 3:271–282. - PubMed
    1. Babst, M., D. Katzmann, W. Snyder, B. Wendland, and S. Emr. 2002. b. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev. Cell. 3:283–289. - PubMed
    1. Bache, K.G., C. Raiborg, A. Mehlum, I.H. Madshus, and H. Stenmark. 2002. Phosphorylation of Hrs downstream of the epidermal growth factor receptor. Eur. J. Biochem. 269:3881–3887. - PubMed
    1. Bache, K.G., A. Brech, A. Mehlum, and H. Stenmark. 2003. a. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. J. Cell Biol. 162:435–442. - PMC - PubMed

Publication types

MeSH terms

Substances