A Role for Ubiquitin Ligase Recruitment in Retrovirus Release

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 24; pp. 13063 - 13068
• Main Authors: Strack, Bettina, Calistri, Arianna, Accola, Molly A., Palu, Giorgio, Gottlinger, Heinrich G.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 21.11.2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Amino Acid Sequence; Avian Sarcoma Viruses - physiology; Biochemistry; Biological Sciences; Budding; Conserved Sequence; Ebola virus; Ebolavirus - physiology; Gag protein; Gene Products, gag - chemistry; Gene Products, gag - metabolism; HeLa Cells; HIV; HIV 1; HIV-1 - physiology; Human immunodeficiency virus; Human immunodeficiency virus 1; Humans; lactacystin; Ligases - metabolism; Mathematical functions; Molecular Sequence Data; Molecules; Polyproteins; Proteins; Recombinant Proteins - metabolism; Rous sarcoma virus; Sequence Alignment; Sequence Homology, Amino Acid; Transfection; ubiquitin ligase; Ubiquitin-Protein Ligases; Ubiquitins; Ubiquitins - metabolism; Virion - physiology; Virus Replication - physiology; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.24.13063

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 p6gagdomain and contains an essential P(T/S)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(T/S)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.