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Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.

Zhadina M, Bieniasz PD - PLoS Pathog. (2010)

Bottom Line: Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins.Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif.These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

View Article: PubMed Central - PubMed

Affiliation: Aaron Diamond AIDS Research Center and Laboratory of Retrovirology, the Rockefeller University, New York, New York, United States of America.

ABSTRACT
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

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Diversity of pathways that can be used to engage the ESCRT machinery.The ESCRT pathway can be engaged by Gag using a variety of natural and artificial mechanisms (i)–(v) that include ubiquitin ligation to trans-acting proteins, (perhaps including the ubiquitin ligase itself) or fusion to Gag. These studies suggest that ubiquitin functions like a transferable L-domain, recruiting class E VPS factors such as ESCRT-I and ALIX, independently of the identity if the protein to which it is attached.
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ppat-1001153-g009: Diversity of pathways that can be used to engage the ESCRT machinery.The ESCRT pathway can be engaged by Gag using a variety of natural and artificial mechanisms (i)–(v) that include ubiquitin ligation to trans-acting proteins, (perhaps including the ubiquitin ligase itself) or fusion to Gag. These studies suggest that ubiquitin functions like a transferable L-domain, recruiting class E VPS factors such as ESCRT-I and ALIX, independently of the identity if the protein to which it is attached.

Mentions: These studies underscore the remarkable flexibility in the ways that the ESCRT pathway can be engaged to achieve viral budding (Fig. 9) Using a single viral Gag protein as a model, particle budding could be achieved by: (i) conventional direct recruitment of the ESCRT pathway via PTAP binding to Tsg101, (ii) direct recruitment of the ESCRT pathway via PPxY binding to a hybrid cofactor consisting of the C2/WW domains of WWP-1 linked to the C-terminal domain of Tsg101, (iii) recruitment of a HECT ubiquitin ligase via a PPxY motif, (iv) recruitment of an isolated HECT domain to a PTAP motif using a hybrid L-domain cofactor consisting of the UEV domain of Tsg101 linked to a HECT domain or (v) direct fusion of ubiquitin to Gag. These results suggest that the cellular factors (in this case Tsg101, ubiquitin ligases and ubiquitin) that are either directly recruited or deposited at the site of viral particle budding behave as modular entities, with domains that are necessary and sufficient for their own recruitment, and distinct domains that are necessary and sufficient for the subsequent recruitment of downstream effectors of particle release (Fig. 9).


Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.

Zhadina M, Bieniasz PD - PLoS Pathog. (2010)

Diversity of pathways that can be used to engage the ESCRT machinery.The ESCRT pathway can be engaged by Gag using a variety of natural and artificial mechanisms (i)–(v) that include ubiquitin ligation to trans-acting proteins, (perhaps including the ubiquitin ligase itself) or fusion to Gag. These studies suggest that ubiquitin functions like a transferable L-domain, recruiting class E VPS factors such as ESCRT-I and ALIX, independently of the identity if the protein to which it is attached.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958808&req=5

ppat-1001153-g009: Diversity of pathways that can be used to engage the ESCRT machinery.The ESCRT pathway can be engaged by Gag using a variety of natural and artificial mechanisms (i)–(v) that include ubiquitin ligation to trans-acting proteins, (perhaps including the ubiquitin ligase itself) or fusion to Gag. These studies suggest that ubiquitin functions like a transferable L-domain, recruiting class E VPS factors such as ESCRT-I and ALIX, independently of the identity if the protein to which it is attached.
Mentions: These studies underscore the remarkable flexibility in the ways that the ESCRT pathway can be engaged to achieve viral budding (Fig. 9) Using a single viral Gag protein as a model, particle budding could be achieved by: (i) conventional direct recruitment of the ESCRT pathway via PTAP binding to Tsg101, (ii) direct recruitment of the ESCRT pathway via PPxY binding to a hybrid cofactor consisting of the C2/WW domains of WWP-1 linked to the C-terminal domain of Tsg101, (iii) recruitment of a HECT ubiquitin ligase via a PPxY motif, (iv) recruitment of an isolated HECT domain to a PTAP motif using a hybrid L-domain cofactor consisting of the UEV domain of Tsg101 linked to a HECT domain or (v) direct fusion of ubiquitin to Gag. These results suggest that the cellular factors (in this case Tsg101, ubiquitin ligases and ubiquitin) that are either directly recruited or deposited at the site of viral particle budding behave as modular entities, with domains that are necessary and sufficient for their own recruitment, and distinct domains that are necessary and sufficient for the subsequent recruitment of downstream effectors of particle release (Fig. 9).

Bottom Line: Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins.Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif.These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

View Article: PubMed Central - PubMed

Affiliation: Aaron Diamond AIDS Research Center and Laboratory of Retrovirology, the Rockefeller University, New York, New York, United States of America.

ABSTRACT
The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

Show MeSH
Related in: MedlinePlus