A role for ubiquitin ligase recruitment in retrovirus release

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(T/S)AP core motif that is widely conserved among lentiviruses, In contrast, the L domains of oncor etroviruses such as Rous sarcoma virus (RSV) have a more N-terminal locatio n and a PPxY core motif. In the present study, we used chimeric Gag constru cts to probe for L domain activity, and observed that the unrelated L domai ns of RSV and HIV-1 both induced the appearance of Gag-ubiquitin conjugates in virus-like particles (VLP). Furthermore, a single-amino acid substituti on that abolished the activity of the RSV L domain in VLP release also abro gated its ability to induce Gag ubiquitination, Particularly robust Gag ubi quitination 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 physiolog ical docking site for the ubiquitin ligase Nedd4. Furthermore, VLP formatio n by a full-length Gag polyprotein was sensitive to lactacystin, which depl etes the levels of free ubiquitin through inhibition of the proteasome, Our findings suggest that the engagement of the ubiquitin conjugation machiner y by L domains plays a crucial role in the release of a diverse group of en veloped viruses.