We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• Klíčová slova
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• MeSH
• buněčné jádro metabolismus   virologie   MeSH
• DEAD-box RNA-helikasy metabolismus   genetika   MeSH
• genom virový MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus * genetika   metabolismus   fyziologie   MeSH
• replikace viru genetika   fyziologie   MeSH
• RNA virová * metabolismus   genetika   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• sestavení viru * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• DHX15 protein, human MeSH Prohlížeč
• RNA virová * MeSH
• RNA-helikasy MeSH

The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.

• Klíčová slova
• Mason-Pfizer monkey virus, endosomes, envelope, intracellular trafficking, transport, virus-like particles,
• MeSH
• AIDS opičí virologie   MeSH
• buněčná membrána metabolismus   virologie   MeSH
• endozomy metabolismus   virologie   MeSH
• genové produkty env genetika   metabolismus   MeSH
• Masonův-Pfizerův opičí virus genetika   fyziologie   MeSH
• sestavení viru MeSH
• transport proteinů MeSH
• transportní vezikuly metabolismus   virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env 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