Abstract
Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.
Highlights
In studying the role of viral and host cell factors involved in retroviral assembly, trafficking, and budding, the mechanism by which capsids are transported through the cytoplasm is still an poorly understood process
Prior data has shown that blocking vesicular trafficking by shifting the temperature of the cell to 20°C causes a significant delay in Mason-Pfizer Monkey Virus (M-PMV) Gag release kinetics and maturation, indicating a role for the cellular vesicular transport system in capsid transport [10,11]
A double mutation, K16A/ K20A, in a basic region of MA, results in the budding of immature capsids into intracellular vesicles, suggesting that these residues play an important role in regulating myristic acid exposure
Summary
In studying the role of viral and host cell factors involved in retroviral assembly, trafficking, and budding, the mechanism by which capsids are transported through the cytoplasm is still an poorly understood process. A single amino acid change from arginine to tryptophan (R55W) in the CTRS causes a switch from B/D type capsid assembly to C-type assembly [3] by preventing nascent Gag molecules from interacting with the dynein light chain Tc-tex and their subsequent transport on microtubules to the pericentriolar region of the cell Another mutation in the matrix (MA) protein of Gag, R22A, displays a transport defect in which assembled immature capsids are unable to reach the plasma membrane resulting in their aggregation under the cortical actin layer. A double mutation, K16A/ K20A, in a basic region of MA, results in the budding of immature capsids into intracellular vesicles, suggesting that these residues play an important role in regulating myristic acid exposure These data and mutants, while providing insights into the dynamics of Gag assembly, intracellular trafficking, and interactions with cellular membranes, have not allowed studies of individual capsid movement within the cell
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