Identification of human VPS37C, a component of endosomal sorting complex required for transport-I important for viral budding
ABSTRACT Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies. In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components. Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein. VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs. In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4. Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag. Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release. Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted. Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I.
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ABSTRACT: Porcine endogenous retrovirus (PERV) is integrated into the genome of all pigs. PERV-A and PERV-B are polytropic and can productively infect human cell lines, while PERV-C is ecotropic. Recombinant PERV-A/C can infect human cells and exhibits high titer replication. Therefore, use of pigs for human xenotransplantation raises concerns about the risks of transfer of this infectious agent from the donors to xenotransplantation recipients. In this study, to establish the strategies to inhibit PERV production from the cells, we investigated the mechanism of PERV budding and anti-PERV activity of Tetherin/BST-2. The results showed that dominant-negative (DN) mutants of WWP-2, Tsg101, and Vps4A/B markedly reduced PERV production in human and porcine cell lines, suggesting that PERV budding utilizes these cellular factors and the cellular multivesicular body (MVB) sorting pathway as well as many other retroviruses. Moreover, PERV production was also reduced by human and porcine Tetherin/BST-2. These data would be useful for developing strategies to inhibit PERV production and may reduce the risk of PERV infection in xenotransplantation.Microbiology and Immunology 06/2014; DOI:10.1111/1348-0421.12166 · 1.31 Impact Factor
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ABSTRACT: ESCRT-0 sorts ubiquitinated EGFR within the early endosome, so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here we employ a suite of software to localise ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whilst ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.Journal of Cell Science 01/2015; DOI:10.1242/jcs.161786 · 5.33 Impact Factor