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Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC
1Laboratory of Molecular Virology, Samsung Biomedical Research Institute, Seoul, Korea.
Abstract:
The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.
Insights
The p4 protein domain of Mason-Pfizer monkey virus (M-PMV) Gag is crucial for capsid assembly. This domain interacts with the chaperonin TRiC, aiding Gag folding and viral capsid formation.
Area of Science:
- Virology
- Molecular Biology
- Protein Folding
Background:
- Mason-Pfizer monkey virus (M-PMV) is a primate type D retrovirus.
- The M-PMV Gag gene encodes a p4 capsid protein domain with an unknown function.
- Chaperonins, like the TCP-1 ring complex (TRiC), assist in cellular protein folding and assembly.
Purpose of the Study:
- To investigate the role of the M-PMV p4 domain in Gag protein stability and capsid assembly.
- To identify potential cellular interactors of the M-PMV p4 domain and Gag polyprotein.
- To elucidate the mechanism by which M-PMV Gag protein folding and assembly occur.
Main Methods:
- Construction and analysis of M-PMV mutants with premature termination codons in the p4 domain.
- Yeast two-hybrid screening to identify protein-protein interactions.
- Cellular co-immunoprecipitation assays to detect Gag-TRiC complex formation.
- ATP hydrolysis assays to assess the dependence of Gag-TRiC association.
Main Results:
- M-PMV mutants lacking parts or all of the p4 domain showed reduced stable Gag protein and capsid assembly.
- The p4 domain specifically interacted with TCP-1gamma, a subunit of the TRiC chaperonin.
- TRiC also associated with M-PMV pp24/16-p12 and HIV p6 domains.
- M-PMV Gag polyprotein associated with TRiC in an ATP-hydrolysis-dependent manner.
- Gag-TRiC association was diminished in p4 truncation mutants.
Conclusions:
- The p4 domain is essential for M-PMV Gag protein stability and efficient capsid assembly.
- Cytosolic chaperonin TRiC plays a critical role in M-PMV Gag folding and/or capsid assembly.
- TRiC likely transiently interacts with nascent Gag molecules to facilitate proper folding, enabling subsequent self-assembly into immature capsids.