Student Theses and Dissertations


Maria Zhadina

Date of Award


Document Type


RU Laboratory

Bieniasz Laboratory


ubiquitin, virus particle budding, retroviral particle release, PFV Gag proteins, HECT domain function


The final stage in the production of infectious retroviral particles is the scission of membrane tethers connecting nascent virions to the host cell. Efficient retrovirus release requires late domains, short peptide motifs within the structural Gag proteins that engage the cellular Class E vacuolar protein sorting (VPS) machinery. Three classes of late domains have been described in retroviruses, each with its own cellular interaction partners: PT/SAP domains bind Tsg101, LxxLF or YPXL domains bind ALIX, and PPxY domains bind Nedd4-like HECT ligases, a class of ubiquitin ligase enzymes. The functional link between HECT ligases and the VPS pathway has not been defined, but multiple lines of evidence suggest that ubiquitin is generally important for late domain activity. Retroviral Gag proteins are often monoubiquitinated, and depletion of free cellular ubiquitin results in accumulation of assembled but immature virions at the cell surface, attached to the cell by membranous stalks. Moreover, mutation of multiple ubiquitin acceptor sites (lysine residues) in HIV-1 and RSV Gag proteins markedly inhibits budding. However, the precise role of ubiquitin in retroviral particle release is poorly understood. To test the importance of direct Gag ubiquitination for viral particle release we constructed a prototype foamy virus (PFV)-derived Gag protein devoid of lysine residues, which can assemble at the plasma membrane in the absence of other viral protein expression. Efficient release of lysine-free PFV Gag virus-like particles (VLPs) required the PSAP late domain and an intact Class E VPS pathway. Remarkably, the MLV PPXY late domain could functionally replace the PSAP motif in lysine-free Gag, retaining its capacity to engage HECT ubiqutin ligases and support particle release. Moreover, overexpression of catalytically active HECT ligases markedly stimulated PPxYdependent lysine-free PFV Gag budding, while catalytically inactive mutants did not. Interestingly, the ability of different HECT domains to promote PFV VLP release correlated with their autoubiquitination activity, but not with their ability to ubiquitinate a Gag substrate containing late domain-proximal lysine residues. Importantly, direct fusion of ubiquitin to the C-terminus of lysine free PFV Gag rescued budding in the absence of conventional late domains. Additionally, ubiquitin fusion dramatically enhanced PSAP-dependent budding. This effect was observable only when a large fraction of Gag carried ubiquitin. Ubiquitin-dependent particle production was abolished by overexpression of dominant negative Vps4 and disruption of the Ile44 hydrophobic patch in ubiquitin. Individual depletion of Tsg101 and ALIX partially suppressed ubiquitin-dependent budding, and the inhibition was enhanced by simultaneous depletion of the two factors. Thus, multiple ubiquitin-binding Class E factors may provide redundant mechanisms for engaging the VPS machinery, which is essential for ubiquitin-dependent budding. Our findings suggest that deposition of ubiquitin at sites of virion assembly by HECT ligases provides docking sites for class E factors, in effect serving as an alternative or additional late domain. However, since both lysine free and ubiquitin fused versions of PFV Gag proteins support budding, the precise nature of the ubiquitin acceptor, be it the Gag protein itself or trans-acting factors, appears unimportant.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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