A role for ubiquitin ligase recruitment in retrovirus release

Abstract

Retroviral Gag polyproteins have specific regions, commonly referred to as late assembly (L) domains, which are required for the efficient separation of assembled virions from the host cell. The L domain of HIV-1 is in the C-terminal p6 gag domain and contains an essential P(T͞S)AP core motif that is widely conserved among lentiviruses. In contrast, the L domains of oncoretroviruses such as Rous sarcoma virus (RSV) have a more N-terminal location and a PPxY core motif. In the present study, we used chimeric Gag constructs to probe for L domain activity, and observed that the unrelated L domains of RSV and HIV-1 both induced the appearance of Gag-ubiquitin conjugates in virus-like particles (VLP). Furthermore, a single-amino acid substitution that abolished the activity of the RSV L domain in VLP release also abrogated its ability to induce Gag ubiquitination. Particularly robust Gag ubiquitination and enhancement of VLP release were observed in the presence of the candidate L domain of Ebola virus, which contains overlapping P(T͞S)AP and PPxY motifs. The release defect of a minimal Gag construct could also be corrected through the attachment of a peptide that serves as a physiological docking site for the ubiquitin ligase Nedd4. Furthermore, VLP formation by a fulllength Gag polyprotein was sensitive to lactacystin, which depletes the levels of free ubiquitin through inhibition of the proteasome. Our findings suggest that the engagement of the ubiquitin conjugation machinery by L domains plays a crucial role in the release of a diverse group of enveloped viruses.

Evidence That HIV Budding in Primary Macrophages Occurs through the Exosome Release Pathway

“…A link between the formation of exosomes and HIV budding in macrophages is supported by recent evidence showing viral budding into intracellular compartments containing MHC II and CD63 and by the presence of these two MVE-associated proteins at high levels on the resulting virion particles within the MVE (16). Previous studies have also shown a requirement for proteins critical to the formation of exosomes in MVEs, such as Tsg-101 (26) and ubiquitin (27), for HIV budding. To date, exosomes from T-cells, Epstein-Barr virus-transformed B-cells, erythrocytes, and dendritic cells have been analyzed (21), but exosomes from primary macrophages have not been studied.…”

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

“…Although one cannot rule out the possibility that K63-linked polyubiquitination might affect these processes indirectly, these data, together with analysis of PRLr degradation in the presence of specific proteasomal inhibitors, raise serious doubts regarding a major role of proteasomes (which usually require K48-linked chains) (10) in the proteolytic fate of PRLr. Interpretation of previously reported effects of proteasome inhibitors on the degradation of either PRLr (29) or erythropoietin/growth hormone receptors (42,45) should include consideration of the possibility of an indirect effect of these inhibitors on the lysosomal pathway, which could be suppressed via depletion of the ubiquitin pool (37). Interestingly, we have never observed a ligandstimulated formation of a PRLr fragment either when following the fate of endogenous PRLr in T47D or 293T cells or when studying recombinant N-terminally HA-tagged or C-terminally Flag-tagged proteins.…”

Distinct Functional Surface Regions on Ubiquitin

“…1A) in Ste2p-Ub3xR were mutated individually or in clusters of 2-4 residues, and yeast strains expressing the mutant chimeras were assayed for their ability to internalize the Ste2p ligand, ␣-factor. Met 1 was not mutated; neither was Ser 28 , since this residue is an alanine in human ubiquitin. All of the receptor-mutant ubiquitin chimeras specifically bound a similar amount of ␣-factor at the cell surface, indicating that the chimeric proteins were transported to the plasma membrane.…”