This effect can be substantially overcome by overexpressing Ub in trans or as a Gag-Ub fusion
This effect can be substantially overcome by overexpressing Ub in trans or as a Gag-Ub fusion. indicate that ubiquitin is actually part of the budding machinery. Ubiquitin (Ub) is a 76-aa protein present in cells either as a free molecule or covalently attached to lysines in a wide variety of proteins. Polyubiquitation of short-lived proteins serves as a tag for proteolysis mediated by the 26S proteasome (1). However, Ub also has other roles in the cell, including one at the plasma membrane, where monoubiquitination of certain receptor proteins promotes their internalization and down-regulation VX-787 (Pimodivir) in a proteasome-independent manner (2C4). The mechanism by which Ub triggers endocytosis of cell surface proteins is currently unclear, but recent work suggests that an endocytic signal within Ub plays a critical role (3, 5). The plasma membrane is also the site of budding for retroviruses, and, 10 years ago, Volker Vogt and his colleagues (6) showed that avian retroviruses contain unexpectedly large amounts of free Ub, amounting to about 100 molecules per virion. This level is 5-fold higher than that of unconjugated Ub in the cytosol, and packaging appears to be specific because other low molecular weight proteins were not detected in the virions. More recently, similar amounts of free Ub have been found in HIV-1, simian immunodeficiency virus, and murine leukemia virus (7). The mechanism by which Ub is packaged into retrovirions is unknown, but it does not involve the viral glycoproteins (Env) because mutants that lack these still contain Ub (6). In some cases (7), a small Rabbit polyclonal to Dcp1a amount (about 30%) of the virion-associated Ub has been found to be conjugated to Gag (Fig. ?(Fig.1),1), the viral protein responsible for particle assembly and budding (8); however, the Ub ligases involved have not been identified, and the significance of Ub for budding has been unknown. Open in a separate window Figure 1 RSV Gag derivatives used in this study. The wild-type polyprotein is shown at the top and its proteolytic cleavage products are indicated. The domains required for budding are indicated below Gag. The M domain mediates the binding of Gag to the cytoplasmic face of the plasma membrane. The I domains provide the major regions of interaction among the 1,500 molecules that create a virion particle. The L domain is required for the virusCcell separation steps that occur late in the budding pathway. The foreign sequences in Gag-GFP, Gag-Ub, T10C-GFP, and T10C-Ub replace the protease (PR) sequence and the last six residues of the nucleocapsid (NC) sequence. In contrast to the role of Ub, a great deal is known about the functions of Gag proteins in virus assembly and budding (8). These proteins (Fig. ?(Fig.1)1) are synthesized on free ribosomes and are subsequently directed to the cytoplasmic face of the plasma membrane by their N-terminal membrane-binding (M) domains. There, approximately 1,500 molecules (9) are packed together into very tight complexes, primarily by means of their interaction (I) domains. The M and I domains lead to the emergence of buds on the surface of the cell, but these are not efficiently released unless the late (L) domain is present. Although the amino acid sequences of M, I, and L are not conserved, these domains are functionally equivalent and exchangeable, even between distantly related viruses. The function provided by L domains is also positionally independent (10). L domains are thought to recruit the cellular machinery needed for virusCcell separation on the plasma membrane. In the case of avian retroviruses, the critical residues of the L domain, PPPPY, are contained within the p2b sequence (Fig. ?(Fig.1)1) and have been shown to be a ligand for WW domains (11C13). A similar sequence has been found in the p12 sequence of murine leukemia virus (14), the pp16 protein of Mason-Pfizer monkey virus (15), and the matrix protein of VX-787 (Pimodivir) rhabdoviruses (16, 17). For HIV-1, the critical residues are PTAP (18, 19), located in the p6 product, and these are possibly involved in binding with an SH3 domain. For equine infectious anemia virus, the critical residues are YPDL in the p9 sequence (20), and these have been shown to bind to and colocalize with adaptor protein (AP)-2, a component of the endocytic machinery on the plasma membrane (21). Interestingly, there appears to be a.Whether these budding factories are unique entities created by Gag proteins or preexisting locations to which Gag proteins are directed remains to be determined. also has other roles in the cell, including one at the plasma membrane, where monoubiquitination of certain receptor proteins promotes their internalization and down-regulation in a proteasome-independent manner (2C4). The mechanism by which Ub triggers endocytosis of cell surface proteins is currently unclear, but recent work suggests that an endocytic signal within Ub plays a critical role (3, 5). The plasma membrane is also the site of budding for retroviruses, and, 10 years ago, Volker Vogt and his colleagues (6) showed that avian retroviruses contain unexpectedly large amounts of free Ub, amounting to about 100 molecules per virion. This level is 5-fold higher than that of unconjugated Ub in the cytosol, and packaging appears to be specific because other low molecular weight proteins were not detected in the virions. More recently, similar amounts of free Ub have been found in HIV-1, simian immunodeficiency virus, and murine leukemia virus (7). The mechanism by which Ub is packaged into retrovirions is unknown, but it does not involve the viral glycoproteins (Env) because mutants that lack these still contain Ub (6). In some cases (7), a small amount (about 30%) of the virion-associated Ub has been found to be conjugated to Gag (Fig. ?(Fig.1),1), the viral protein responsible for particle assembly and budding (8); however, the Ub ligases involved have not been identified, and the significance of Ub for budding has been unknown. Open in a separate window Figure 1 RSV Gag derivatives used in this study. The wild-type polyprotein is shown at the top and its proteolytic cleavage products are indicated. The domains required for budding are indicated below Gag. The M domain mediates the binding of Gag to the cytoplasmic face of the plasma membrane. The I domains provide the major regions of interaction among the 1,500 molecules that create a virion particle. The L domain is required for the virusCcell separation steps that occur late in the budding pathway. The foreign sequences in Gag-GFP, Gag-Ub, T10C-GFP, and T10C-Ub change the protease (PR) sequence and the last six residues of the nucleocapsid (NC) sequence. In contrast to the part of Ub, a great deal is known about the functions of Gag proteins in virus assembly and budding (8). These proteins (Fig. ?(Fig.1)1) are synthesized about free ribosomes and are subsequently directed to the cytoplasmic face of the plasma membrane by their N-terminal membrane-binding (M) domains. There, approximately 1,500 molecules (9) are packed together into very tight complexes, primarily by means of their connection (I) domains. The M and I domains lead to the emergence of buds on the surface of the cell, but these are not efficiently released unless the late (L) website is present. Even though amino VX-787 (Pimodivir) acid sequences of M, I, and L are not conserved, these domains are functionally equal and exchangeable, actually between distantly related viruses. The function provided by L domains is also positionally self-employed (10). L domains are thought to recruit the cellular machinery needed for virusCcell separation within the plasma membrane. In the case of avian retroviruses, the essential residues of the L website, PPPPY, are contained within the p2b sequence (Fig. ?(Fig.1)1) and have been shown to be a ligand for WW domains (11C13). A similar sequence has been found in the p12 sequence of murine leukemia disease (14), the pp16 protein of Mason-Pfizer monkey disease (15), and the matrix protein of rhabdoviruses (16, 17). For HIV-1, the essential residues are PTAP (18, 19), located in the p6 product, and these are possibly involved in binding with an SH3 website. For equine infectious anemia disease, the essential residues are YPDL in the p9 sequence (20), and these have been shown to bind to and colocalize with adaptor protein (AP)-2, a component of the endocytic machinery within the plasma membrane (21). Interestingly, there appears to be a correlation between L domains and Ub. In those viruses where Ub has been found to be linked to Gag, the conjugated lysines are in very close proximity to the L website (7), and ubiquitination of Gag proteins has.