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 p6gag domain 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 full-length 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.

Ubiquitin is part of the retrovirus budding machinery

Retroviruses contain relatively large amounts of ubiquitin, but the significance of this finding has been unknown. Here, we show that drugs that are known to reduce the level of free ubiquitin in the cell dramatically reduced the release of Rous sarcoma virus, an avian retrovirus. This effect was suppressed by overexpressing ubiquitin and also by directly fusing ubiquitin to the C terminus of Gag, the viral protein that directs budding and particle release. The block to budding was found to be at the plasma membrane, and electron microscopy revealed that the reduced level of ubiquitin results in a failure of mature virus particles to separate from each other and from the plasma membrane during budding. These data indicate that ubiquitin is actually part of the budding machinery.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.