The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.

• Klíčová slova
• Anterograde transport, Cytoskeleton, Envelope, Gag, Live cell-imaging, M-PMV,
• MeSH
• AIDS opičí metabolismus   virologie   MeSH
• buněčná membrána virologie   MeSH
• Cercopithecus aethiops MeSH
• genové produkty env genetika   metabolismus   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• Macaca mulatta MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• genové produkty env MeSH
• genové produkty gag MeSH

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.

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• genetické vektory genetika   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• kapsida metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   fyziologie   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• molekulární zobrazování MeSH
• pohyb MeSH
• proviry genetika   metabolismus   fyziologie   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• sestavení viru MeSH
• transport proteinů MeSH
• viabilita buněk MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fluorescenční barviva MeSH
• genové produkty gag MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH