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Eric Hunter - One of the best experts on this subject based on the ideXlab platform.
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A MasonPfizer monkey virus Gag-GFP fusion vector allows visualization of capsid transport in live cells and demonstrates a role for microtubules. PLoS One
IN PRESS, 2014Co-Authors: Jasmine Clark, Petra Grznarova, Tomáš Ruml, Paul Spearman, Jan Lipov, Eric HunterAbstract: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 usefu
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A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules
PloS one, 2013Co-Authors: Jasmine Clark, Petra Grznarova, Tomáš Ruml, Paul Spearman, Jan Lipov, Elizabeth Stansell, William E. Diehl, Eric HunterAbstract: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.
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Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules.
Virology, 2013Co-Authors: Lara E. Pereira, Jasmine Clark, Petra Grznarova, Xiaoyun Wen, Rachel Lacasse, Tomáš Ruml, Paul Spearman, Eric HunterAbstract: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 4 h 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.
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The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a Pericentriolar Region of the cell.
Traffic (Copenhagen Denmark), 2003Co-Authors: Jeffrey N. Sfakianos, Rachel A. Lacasse, Eric HunterAbstract:Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the Pericentriolar Region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in Pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this Region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the Pericentriolar assembly site requires the env-gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates Pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.
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M-PMV capsid transport is mediated by Env/Gag interactions at the Pericentriolar recycling endosome
Traffic (Copenhagen Denmark), 2003Co-Authors: Jeffrey N. Sfakianos, Eric HunterAbstract:Cytoplasmic transport of Gag molecules to the site of budding is an important but poorly understand process in retroviral assembly. Our previous studies of Mason-Pfizer monkey virus showed that, for this retrovirus, Gag is assembled into capsids at a Pericentriolar Region and that Env is necessary for efficient transport out of the site. An Env requirement for cytoplasmic transport implicates vesicular trafficking in this process even though the capsids remain cytoplasmic and do not bud into intracellular compartments in the cells studied to date. We show here that the secretory pathway of the cell is not directly involved in Gag transport since the latter was not inhibited by BFA, nor did Gag colocalize with markers of the ER, Golgi, or TGN. Instead, colocalization was observed between Gag and endocytosed transferrin and with Rab11, suggesting that Pericentriolar recycling endosomes play a critical role in this process. Mutants of Rab11 that inhibit efflux of transferrin from the recycling endosome also inhibited Gag transport. Our studies show that Env colocalizes with Gag at the Pericentriolar assembly site, and provide evidence that Env must travel through this compartment in order to initiate export of the capsids from the site of assembly. Thus, for the first time, endocytic trafficking of a retroviral Env glycoprotein is linked to the efficient cytoplasmic transport of Gag.
Catherine Angenieux - One of the best experts on this subject based on the ideXlab platform.
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birbeck granule like organized smooth endoplasmic reticulum resulting from the expression of a cytoplasmic yfp tagged langerin
PLOS ONE, 2013Co-Authors: C Lenormand, Bertrand Cinquin, Sabine Bardin, Catherine Angenieux, Huguette Bausinger, Coralie SpiegelhalterAbstract:Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the Pericentriolar Region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of “Organized Smooth Endoplasmic Reticulum” (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a “double-lock” mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.
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A monomerizing substitution in YFP impairs the formation of BG-like OSER and restores BG dynamics.
2013Co-Authors: C Lenormand, Bertrand Cinquin, Sabine Bardin, Catherine Angenieux, Huguette Bausinger, Coralie Spiegelhalter, Anita Eckly, Fabienne Proamer, David Wall, Ben LichAbstract:(A) M10 cells stably expressing mYFP-Lang were fixed and the YFP distribution was examined by confocal microscopy. Overlays of 5–10 z-sections from different representative cells are depicted. Left and middle panels: images representative of the majority of cells, with a Pericentriolar concentration of fluorescence and small vesicles dispersed across the cytoplasm. Right panel: YFP+ puncta present only in a minority of cells. Scale bars: 25 µm. (B) FRAP experiments were carried out as in Fig. 5B. Left panel: to study the fluorescence recovery in the Pericentriolar Region, 16 cells were analyzed for each plot. Right panel: to study the fluorescence recovery in enlarged vesicles, 8 cells were analyzed for each plot. Error bars represent the standard error of the mean. Fi and Fm indicate the immobile and mobile fractions of mYFP-Lang molecules, respectively. (C) Soluble membrane extracts (30 µg) of M10 cells stably expressing transgenic Lang-YFP, YFP-Lang or mYFP-Lang were digested or not (NT) with PNGaseF (F) or endoglycosidase Hf (H) and separated by 7.5% SDS-PAGE. Fusion proteins were revealed by western blotting with an anti-GFP antibody. R and S indicate endoglycosidase Hf-resistant and sensitive species, respectively. Untransfected cells (WT) were used as a control.
Maxine L. Linial - One of the best experts on this subject based on the ideXlab platform.
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Foamy Virus Assembly with Emphasis on Pol Encapsidation
Viruses, 2013Co-Authors: Eun Gyung Lee, Carolyn R. Stenbak, Maxine L. LinialAbstract:Foamy viruses (FVs) differ from all other genera of retroviruses (orthoretroviruses) in many aspects of viral replication. In this review, we discuss FV assembly, with special emphasis on Pol incorporation. FV assembly takes place intracellularly, near the Pericentriolar Region, at a site similar to that used by betaretroviruses. The Regions of Gag, Pol and genomic RNA required for viral assembly are described. In contrast to orthoretroviral Pol, which is synthesized as a Gag-Pol fusion protein and packaged through Gag-Gag interactions, FV Pol is synthesized from a spliced mRNA lacking all Gag sequences. Thus, encapsidation of FV Pol requires a different mechanism. We detail how WT Pol lacking Gag sequences is incorporated into virus particles. In addition, a mutant in which Pol is expressed as an orthoretroviral-like Gag-Pol fusion protein is discussed. We also discuss temporal regulation of the protease, reverse transcriptase and integrase activities of WT FV Pol.
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foamy virus capsid assembly occurs at a Pericentriolar Region through a cytoplasmic targeting retention signal in gag
Traffic, 2006Co-Authors: Scott W. Eastman, Maxine L. LinialAbstract:Foamy viruses (FV) are unusual retroviruses that differ in many aspects of their life cycle from the orthoretroviruses such as human immunodeficiency virus. Similar to Mason-Pfizer monkey virus (MPMV), FV assemble into capsids intracellularly. The capsids are then transported to a cellular membrane for acquisition of envelope (Env) glycoproteins and budding. However, unlike MPMV, budding of FV is dependent upon the presence of Env. Previous work suggested that FV Env proteins are localized to the endoplasmic reticulum (ER) where budding takes place. However, very little was known about the details of FV assembly. We have used immunofluorescence and electron microscopy to visualize the intracellular location of FV assembly and budding. We have found that, as in the case of MPMV, FV capsids assemble at a Pericentriolar site in the cytoplasm. Surprisingly, FV Env is mostly absent from this site and, contrary to expectations, FV capsid structural protein (Gag) is absent from the ER. Gag and Env only co-localize at the trans-Golgi network, suggesting that Env-Gag interactions that are required for viral egress from the cell, occurs at this site. Finally, inhibitor studies suggest an important role of microtubule networks for foamy viral assembly and budding.
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Foamy Virus Capsid Assembly Occurs at a Pericentriolar Region Through a Cytoplasmic Targeting/Retention Signal in Gag
Traffic (Copenhagen Denmark), 2006Co-Authors: Scott W. Eastman, Maxine L. LinialAbstract:Foamy viruses (FV) are unusual retroviruses that differ in many aspects of their life cycle from the orthoretroviruses such as human immunodeficiency virus. Similar to Mason-Pfizer monkey virus (MPMV), FV assemble into capsids intracellularly. The capsids are then transported to a cellular membrane for acquisition of envelope (Env) glycoproteins and budding. However, unlike MPMV, budding of FV is dependent upon the presence of Env. Previous work suggested that FV Env proteins are localized to the endoplasmic reticulum (ER) where budding takes place. However, very little was known about the details of FV assembly. We have used immunofluorescence and electron microscopy to visualize the intracellular location of FV assembly and budding. We have found that, as in the case of MPMV, FV capsids assemble at a Pericentriolar site in the cytoplasm. Surprisingly, FV Env is mostly absent from this site and, contrary to expectations, FV capsid structural protein (Gag) is absent from the ER. Gag and Env only co-localize at the trans-Golgi network, suggesting that Env-Gag interactions that are required for viral egress from the cell, occurs at this site. Finally, inhibitor studies suggest an important role of microtubule networks for foamy viral assembly and budding.
C Lenormand - One of the best experts on this subject based on the ideXlab platform.
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birbeck granule like organized smooth endoplasmic reticulum resulting from the expression of a cytoplasmic yfp tagged langerin
PLOS ONE, 2013Co-Authors: C Lenormand, Bertrand Cinquin, Sabine Bardin, Catherine Angenieux, Huguette Bausinger, Coralie SpiegelhalterAbstract:Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the Pericentriolar Region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of “Organized Smooth Endoplasmic Reticulum” (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a “double-lock” mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.
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A monomerizing substitution in YFP impairs the formation of BG-like OSER and restores BG dynamics.
2013Co-Authors: C Lenormand, Bertrand Cinquin, Sabine Bardin, Catherine Angenieux, Huguette Bausinger, Coralie Spiegelhalter, Anita Eckly, Fabienne Proamer, David Wall, Ben LichAbstract:(A) M10 cells stably expressing mYFP-Lang were fixed and the YFP distribution was examined by confocal microscopy. Overlays of 5–10 z-sections from different representative cells are depicted. Left and middle panels: images representative of the majority of cells, with a Pericentriolar concentration of fluorescence and small vesicles dispersed across the cytoplasm. Right panel: YFP+ puncta present only in a minority of cells. Scale bars: 25 µm. (B) FRAP experiments were carried out as in Fig. 5B. Left panel: to study the fluorescence recovery in the Pericentriolar Region, 16 cells were analyzed for each plot. Right panel: to study the fluorescence recovery in enlarged vesicles, 8 cells were analyzed for each plot. Error bars represent the standard error of the mean. Fi and Fm indicate the immobile and mobile fractions of mYFP-Lang molecules, respectively. (C) Soluble membrane extracts (30 µg) of M10 cells stably expressing transgenic Lang-YFP, YFP-Lang or mYFP-Lang were digested or not (NT) with PNGaseF (F) or endoglycosidase Hf (H) and separated by 7.5% SDS-PAGE. Fusion proteins were revealed by western blotting with an anti-GFP antibody. R and S indicate endoglycosidase Hf-resistant and sensitive species, respectively. Untransfected cells (WT) were used as a control.
Tomáš Ruml - One of the best experts on this subject based on the ideXlab platform.
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Interaction of Mason-Pfizer monkey virus matrix protein with plasma membrane
Frontiers in microbiology, 2014Co-Authors: Jan Prchal, Tomáš Ruml, Tomáš Kroupa, Richard HrabalAbstract:Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane through its N-terminal domain, the matrix protein. However, single generas of Retroviridae family differ in the way how they interact with plasma membrane. While in case of lentiviruses (e.g. human immunodeficiency virus (HIV)) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, betaretroviruses (Mason-Pfizer monkey virus (M-PMV)) first assemble their virus-like particles in the Pericentriolar Region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV matrix protein with plasma membrane with emphasis on the structural aspects of the interaction with single phospholipids.
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Reviewed by:
2014Co-Authors: Jan Prchal, Tomáš Ruml, Tomáš Kroupa, Richard Hrabal, Atsushi KoitoAbstract:Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane (PM) through its N-terminal domain, the matrix protein (MA). However, single genera of Retroviridae family differ in the way how they interact with PM. While in case of Lentiviruses (e.g., human immunodeficiency virus) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, Betaretroviruses [Mason-Pfizer monkey virus (M-PMV)] first assemble their virus-like particles (VLPs) in the Pericentriolar Region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV MA with PM with emphasis on the structural aspects of the interaction with single phospholipids
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A MasonPfizer monkey virus Gag-GFP fusion vector allows visualization of capsid transport in live cells and demonstrates a role for microtubules. PLoS One
IN PRESS, 2014Co-Authors: Jasmine Clark, Petra Grznarova, Tomáš Ruml, Paul Spearman, Jan Lipov, Eric HunterAbstract: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 usefu
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A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules
PloS one, 2013Co-Authors: Jasmine Clark, Petra Grznarova, Tomáš Ruml, Paul Spearman, Jan Lipov, Elizabeth Stansell, William E. Diehl, Eric HunterAbstract: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.
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Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules.
Virology, 2013Co-Authors: Lara E. Pereira, Jasmine Clark, Petra Grznarova, Xiaoyun Wen, Rachel Lacasse, Tomáš Ruml, Paul Spearman, Eric HunterAbstract: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 4 h 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.