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

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.

Quantitative Super-Resolution Imaging Reveals Protein Stoichiometry and Nanoscale Morphology of Assembling HIV-Gag Virions

“…For comparison with previous measurements of fully formed virions, we examined the largest 10% of clusters, and found an average diameter of 166 +/-26 nm for Gag-mEos2 and 302 +/-90 nm for Gag-tdEos. For Gag-mEos2 this is in good agreement with EM of budded VLPs, reported in different studies to be 100-200 nm or 145 +/-25 nm in size 5,19 . It moreover indicates that the GagtdEos particles are unusually large.…”

“…In support of this, we estimate based on our measurements of nascent virion size and protein number that the Gag-tdEos forms a patchy lattice covering only ~10-20% of the virion surface, based on the lattice spacing measured by EM. As further evidence, a separate study of elongated Gag proteins revealed an increase in virion size and a discontinuous density of Gag 19 . However, our data allow us to go further, in showing that these differences in packing exist throughout most of the assembly process.…”

“…Performing our sector based analysis on HIV-Gag clusters also revealed striking differences between the sizes of assembling virions formed with Gag-mEos2 or Gag-tdEos ( Figure 3A, B, C). The average radius was 53 +/-12 nm for Gag-mEos2 5,19 versus 86 +/-26 nm for Gag-tdEos ( Figure 3C), differing by a factor of 1.6. For comparison with previous measurements of fully formed virions, we examined the largest 10% of clusters, and found an average diameter of 166 +/-26 nm for Gag-mEos2 and 302 +/-90 nm for Gag-tdEos.…”

Macrophages and HIV-1: dangerous liaisons

“…Gag drives HIV-1 budding through the Gag p6 late domain (L domain). L domain binds the tumour susceptibility gene 101 (TSG101), hijacking the host's MVB vesicle formation machinery, and recruits proteins of the ESCRT (endosomal sorting complexes required for transport) family, which regulate MVB biogenesis and cellular vacuolar protein sorting pathways (Pornillos et al, 2003;Strack et al, 2003;von Schwedler et al, 2003). The release of HIV-1 also seems to differ in T cells and in macrophages.…”

Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding

“…In addition to Vps28, a component of yeast and mammalian ESCRT-I (Katzmann et al 2001;Martin-Serrano et al 2003), an as-yet-unidentified mammalian ortholog of yeast Vps37 likely complements the action of Tsg101. Recent reports identified two additional partners of Tsg101, namely, AIP1, the ortholog of Bro1 (Strack et al 2003;von Schwedler et al 2003), and Hrs (Bache et al 2003a;Katzmann et al 2003;Pornillos et al 2003). In an effort to resolve the sorting action of Tsg101, we screened cDNA libraries for Tsg101-associated proteins by using the yeast two-hybrid system.…”