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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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Synergy between a PSAP motif and ubiquitin during viral budding and effect of Gag-ubiquitin levels on particle release.(A) Schematic representation of some examples of the Lck-Gag proteins used in these experiments, encoding PSAP and/or PPxY L-domains and a single ubiquitin moiety fused at the carboxyl-terminus. (B) VLP release from by 293T cells expressing Lck-Gag proteins containing the indicated L-domains that were present alone or in combination with each other and/or directly fused ubiquitin (see panel A), measured by quantitative western blotting. The chart to the right of the blot shows the yield of VLPs (mean+SD of 2 experiments) and values are presented relative to the VLP yield obtained in the presence of both L-domains and ubiquitin (Lck-Gag(PSAP)-PY-Ub), which was assigned a value of 1. (C) VLP production from 293T cells expressing Lck-Gag(L-) or Lck-Gag(PSAP) proteins, where the total amount of Gag protein expressed was constant, but varying proportions carried a fused ubiquitin. Cells were transfected with equal total amounts of Lck-Gag(L-)+Lck-Gag-Ub (left panels) or Lck-Gag(PSAP)+Lck-Gag(PSAP)-Ub expression plasmids, and the indicated fraction of the total transfected plasmid mixture expressed the ubiquitin fused form of the Gag protein. VLPs were harvested both at 24h and 48h after transfection and subjected to western blot analysis with αPFV antiserum.
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ppat-1001153-g006: Synergy between a PSAP motif and ubiquitin during viral budding and effect of Gag-ubiquitin levels on particle release.(A) Schematic representation of some examples of the Lck-Gag proteins used in these experiments, encoding PSAP and/or PPxY L-domains and a single ubiquitin moiety fused at the carboxyl-terminus. (B) VLP release from by 293T cells expressing Lck-Gag proteins containing the indicated L-domains that were present alone or in combination with each other and/or directly fused ubiquitin (see panel A), measured by quantitative western blotting. The chart to the right of the blot shows the yield of VLPs (mean+SD of 2 experiments) and values are presented relative to the VLP yield obtained in the presence of both L-domains and ubiquitin (Lck-Gag(PSAP)-PY-Ub), which was assigned a value of 1. (C) VLP production from 293T cells expressing Lck-Gag(L-) or Lck-Gag(PSAP) proteins, where the total amount of Gag protein expressed was constant, but varying proportions carried a fused ubiquitin. Cells were transfected with equal total amounts of Lck-Gag(L-)+Lck-Gag-Ub (left panels) or Lck-Gag(PSAP)+Lck-Gag(PSAP)-Ub expression plasmids, and the indicated fraction of the total transfected plasmid mixture expressed the ubiquitin fused form of the Gag protein. VLPs were harvested both at 24h and 48h after transfection and subjected to western blot analysis with αPFV antiserum.

Mentions: Next we analyzed the effect of combining L-domains and ubiquitin on VLP release. To accomplish this, Lck-Gag proteins containing various combinations of the L-domains and C-terminally fused ubiquitin (Fig. 6A) were expressed. Quantitative analyses revealed that directly fused ubiquitin-dependent (Lck-Gag-Ub) particle release was at least as efficient as that driven by PSAP (Lck-Gag(PSAP)) or PPxY (Lck-Gag-PY) L-domains (Fig. 6B). Moreover, and in contrast to the previous report with EIAV Gag [48], we found that the combined presence of fused ubiquitin and a PSAP L-domain (in Lck-Gag(PSAP)-Ub) resulted in strongly synergistic effects on particle release (Fig. 6B). Specifically, Lck-Gag(PSAP)-Ub generated ∼20-fold and ∼6-fold more particles than Lck-Gag(PSAP) and Lck-Gag-Ub, respectively (Fig. 6B). No such synergy was observed when a PPxY L-domain and ubiquitin were combined in the same Gag protein. In fact, the Lck-Gag-Ub and the Lck-Gag-PY-Ub generated extracellular particles with approximately the same efficiency (Fig. 6B). Less dramatic, but nonetheless synergistic enhancement of particle release was evident when PPxY and PSAP motifs were both present (in the absence of ubiquitin fusion, Fig. 6B). In this case, the presence of the PPxY motif (in Lck-Gag(PSAP)-PY) enhanced particle release approximately ∼5-fold as compared to the situation where the PSAP motif was the only L-domain (in Lck-Gag(PSAP), Fig. 6B). Overall these results are consistent with the notion that ubiquitin behaves essentially like an L-domain, and further suggests that it functions synergistically with a PT/SAP motif, and redundantly with a PPxY motif.


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

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

Synergy between a PSAP motif and ubiquitin during viral budding and effect of Gag-ubiquitin levels on particle release.(A) Schematic representation of some examples of the Lck-Gag proteins used in these experiments, encoding PSAP and/or PPxY L-domains and a single ubiquitin moiety fused at the carboxyl-terminus. (B) VLP release from by 293T cells expressing Lck-Gag proteins containing the indicated L-domains that were present alone or in combination with each other and/or directly fused ubiquitin (see panel A), measured by quantitative western blotting. The chart to the right of the blot shows the yield of VLPs (mean+SD of 2 experiments) and values are presented relative to the VLP yield obtained in the presence of both L-domains and ubiquitin (Lck-Gag(PSAP)-PY-Ub), which was assigned a value of 1. (C) VLP production from 293T cells expressing Lck-Gag(L-) or Lck-Gag(PSAP) proteins, where the total amount of Gag protein expressed was constant, but varying proportions carried a fused ubiquitin. Cells were transfected with equal total amounts of Lck-Gag(L-)+Lck-Gag-Ub (left panels) or Lck-Gag(PSAP)+Lck-Gag(PSAP)-Ub expression plasmids, and the indicated fraction of the total transfected plasmid mixture expressed the ubiquitin fused form of the Gag protein. VLPs were harvested both at 24h and 48h after transfection and subjected to western blot analysis with αPFV antiserum.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958808&req=5

ppat-1001153-g006: Synergy between a PSAP motif and ubiquitin during viral budding and effect of Gag-ubiquitin levels on particle release.(A) Schematic representation of some examples of the Lck-Gag proteins used in these experiments, encoding PSAP and/or PPxY L-domains and a single ubiquitin moiety fused at the carboxyl-terminus. (B) VLP release from by 293T cells expressing Lck-Gag proteins containing the indicated L-domains that were present alone or in combination with each other and/or directly fused ubiquitin (see panel A), measured by quantitative western blotting. The chart to the right of the blot shows the yield of VLPs (mean+SD of 2 experiments) and values are presented relative to the VLP yield obtained in the presence of both L-domains and ubiquitin (Lck-Gag(PSAP)-PY-Ub), which was assigned a value of 1. (C) VLP production from 293T cells expressing Lck-Gag(L-) or Lck-Gag(PSAP) proteins, where the total amount of Gag protein expressed was constant, but varying proportions carried a fused ubiquitin. Cells were transfected with equal total amounts of Lck-Gag(L-)+Lck-Gag-Ub (left panels) or Lck-Gag(PSAP)+Lck-Gag(PSAP)-Ub expression plasmids, and the indicated fraction of the total transfected plasmid mixture expressed the ubiquitin fused form of the Gag protein. VLPs were harvested both at 24h and 48h after transfection and subjected to western blot analysis with αPFV antiserum.
Mentions: Next we analyzed the effect of combining L-domains and ubiquitin on VLP release. To accomplish this, Lck-Gag proteins containing various combinations of the L-domains and C-terminally fused ubiquitin (Fig. 6A) were expressed. Quantitative analyses revealed that directly fused ubiquitin-dependent (Lck-Gag-Ub) particle release was at least as efficient as that driven by PSAP (Lck-Gag(PSAP)) or PPxY (Lck-Gag-PY) L-domains (Fig. 6B). Moreover, and in contrast to the previous report with EIAV Gag [48], we found that the combined presence of fused ubiquitin and a PSAP L-domain (in Lck-Gag(PSAP)-Ub) resulted in strongly synergistic effects on particle release (Fig. 6B). Specifically, Lck-Gag(PSAP)-Ub generated ∼20-fold and ∼6-fold more particles than Lck-Gag(PSAP) and Lck-Gag-Ub, respectively (Fig. 6B). No such synergy was observed when a PPxY L-domain and ubiquitin were combined in the same Gag protein. In fact, the Lck-Gag-Ub and the Lck-Gag-PY-Ub generated extracellular particles with approximately the same efficiency (Fig. 6B). Less dramatic, but nonetheless synergistic enhancement of particle release was evident when PPxY and PSAP motifs were both present (in the absence of ubiquitin fusion, Fig. 6B). In this case, the presence of the PPxY motif (in Lck-Gag(PSAP)-PY) enhanced particle release approximately ∼5-fold as compared to the situation where the PSAP motif was the only L-domain (in Lck-Gag(PSAP), Fig. 6B). Overall these results are consistent with the notion that ubiquitin behaves essentially like an L-domain, and further suggests that it functions synergistically with a PT/SAP motif, and redundantly with a PPxY motif.

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