Ux used subcellular fractionation techniques to examine the localization of Gag
Ux used subcellular fractionation techniques to examine the localization of Gag in transfected 293T cells and chronically infected MOLT Tcells. Cell lysates prepared from virus-expressing cells were fractionated on iodixanol gradients. The sedimentation of Gag was compared to that of markers for plasma membrane, late endosomes, small vesicles, and soluble proteins [46]. The results indicated that Gag was found in fractions that corresponded to both the plasma membrane and late endosomes. Genomic RNA was observed primarily in late endosomal and soluble fractions. Similar results were obtained in the MOLT T cells. These observations, which were confirmed by immunofluorescence and electron microscopy, led the authors to conclude that HIV-1 assembly in 293T and chronically infected T cells takes place at both the plasma membrane and in MVBs. Removal of the zinc fingers in the NC domain resulted in a shift in Gag localization from endosomal to recycling vesicle fractions, and a loss in the cosedimentation of Gag and genomic RNA.In his keynote address, Wesley Sundquist discussed several aspects of the cell biology and biochemistry of HIV-1 budding. He first described some of the cellular apparatus that associates with Tsg101. In addition to the ESCRT-1 components Vps28 and Vps37, Tsg101 also binds Hrs, Alix, the GGA proteins, and TOM1L1. Interestingly, TOM1L1 also interacts with Nedd4-like E3 ubiquitin ligases, raising the possibility that it might play a role in the recruitment of PPxY-containing retroviruses into the MVB pathway. Ubiquitination of cargo proteins is often (but not always) required for their sorting into MVBs, and there are several lines of evidence suggesting that ubiquitination of Gag PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27324125 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27532042 itself may play a positive role in virus release. A number of components of the MVB machinery, including Hrs, Tsg101, and the ESCRT-II component EAP45, contain motifs that directly bind ubiquitin. HIV-1 Gag, for example, could interact with Tsg101 not only through its P(T/S)AP motif in p6 but also through ubiquitin moieties attached to several domains of Gag [50]. Purification and analysis of ESCRT-I complexes in Sundquist’s lab revealed a heretofore unrecognized fourth component of ESCRT-I, referred to as EI4A (and variant EI4B). Finally, Sundquist described his lab’s Olumacostat glasaretil biological activity studies on Alix. While it is now fairly clear that Alix is the major latedomain-interacting protein for EIAV, as mentioned above HIV-1 p6 also interacts with Alix. A role for Alix in HIV-1 release is most apparent when the Gag/Tsg101 interaction has been abolished. Structural studies with the central, Gag-binding domain of Alix revealed a V-shaped fold, with the YPxnL binding site lying inside the base of the V. The continuing discovery of additional components of ESCRT and associated machinery adds to the complexity of the endosomal sorting (and virus budding) machinery. Sundquist pointed out that 100 proteins are involved in endocytosis and that a comparable number of proteins may ultimately be implicated in MVB biogenesis. It will be of great interest to define which of this multitude of cellular factors are required for the release of HIV-1 and other retroviruses. Previous studies from the lab of Jaisri Lingappa demonstrated that HIV-1 assembly proceeds through the formation of a series of discrete intermediates of 10S, 80S, 150S, and 500S, culminating in a 750S immature VLP [51]. The subcellular localization of these assembly intermediates was investigated by Lingappa and.