A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules

Clark, Jasmine; Grznarova, Petra; Stansell, Elizabeth; Diehl, William E.; Lipov, Jan; Spearman, Paul; Ruml, Tomáš; Hunter, Eric

doi:10.1371/journal.pone.0083863

Cited by 10 publications

(15 citation statements)

References 32 publications

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“…2). As we described previously (Clark et al, in press), bidirectional movement and occasional stalling of Gag–GFP particles was often noted, which is typical for microtubule motor-based movement given that switching of cargo between minus- and plus-ended motors tends to occur (Hendricks et al, 2010; Ross et al, 2008; Shah et al, 2000). These data are thus consistent with the pulse-chase experiments suggesting a role for microtubules in capsid transport.…”

Section: Resultssupporting

confidence: 53%

“…An advantage of pulse-chase time course assays is that Gag proteins can be tracked from the stages of synthesis and assembly to the point of release. Furthermore, since M-PMV capsid movement can be bidirectional, which we have detailed by live imaging (Clark et al, in press), pulse-chase assays ensure that the net direction of intracellular M-PMV capsid transport being studied is indeed anterograde. As indicated by the experimental outline in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Live cell microscopy experiments were therefore performed to gain a real time perspective of capsid and Env movements before and during nocodazole treatment. Using live-cell microscopy, M-PMV-producing CMMT cells were visualized in real time approximately 7 h post-transfection with a plasmid construct expressing codon-optimized M-PMV Gag–GFP (pSARM–GagGFP–M100A; Clark et al, in press). Analysis at this early time point allows for a balance between the level of detectable GFP signal and the number of Gag– GFP particles that is statistically relevant for velocity measurements.…”

Section: Resultsmentioning

confidence: 99%

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Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules

et al. 2014

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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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Section: Resultssupporting

confidence: 53%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules

et al. 2014

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“…All OMEGA algorithmic components were tested on artificial image and trajectory data as described in Supplemental Information 1 and elsewhere (Rigano et al, 2018). As proof of concept, OMEGA supported motion analysis workflows were utilized to analyzed both standard SPT benchmarking datasets (Figure 7;Chenouard et al, 2014), as well as real-life datasets depicting retroviral particle trafficking within living human cells (Clark et al, 2013;Pereira et al, 2012).…”

Section: Statement Of Purpose: Integration Of Particle Tracking Work-mentioning

confidence: 99%

OMEGA: a software tool for the management, analysis, and dissemination of intracellular trafficking data that incorporates motion type classification and quality control

Rigano

Galli

Clark

et al. 2018

Preprint

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MOTIVATIONParticle tracking coupled with time-lapse microscopy is critical for understanding the dynamics of intracellular processes of clinical importance. Spurred on by advances in the spatiotemporal resolution of microscopy and automated computational methods, this field is increasingly amenable to multi-dimensional high-throughput data collection schemes (Snijder et al., 2012). Typically, complex particle tracking datasets generated by individual laboratories are produced with incompatible methodologies that preclude comparison to each other. There is therefore an unmet need for data management systems that facilitate data standardization, meta-analysis, and structured data dissemination. The integration of analysis, visualization, and quality control capabilities into such systems would eliminate the need for manual transfer of data to diverse downstream analysis tools. At the same time, it would lay the foundation for shared trajectory data, particle tracking, and motion analysis standards.RESULTSHere, we present Open Microscopy Environment inteGrated Analysis (OMEGA), a cross-platform data management, analysis, and visualization system, for particle tracking data, with particular emphasis on results from viral and vesicular trafficking experiments. OMEGA provides intuitive graphical interfaces to implement integrated particle tracking and motion analysis workflows while providing easy to use facilities to automatically keep track of error propagation, harvest data provenance and ensure the persistence of analysis results and metadata. Specifically, OMEGA: 1) imports image data and metadata from data management tools such as the Open Microscopy Environment Remote Objects (OMERO; Allan et al., 2012); 2) tracks intracellular particles movement; 3) facilitates parameter optimization and trajectory results inspection and validation; 4) performs downstream trajectory analysis and motion type classification; 5) estimates the uncertainty propagating through the motion analysis pipeline; and, 6) facilitates storage and dissemination of analysis results, and analysis definition metadata, on the basis of our newly proposed FAIRsharing.org complainant Minimum Information About Particle Tracking Experiments (MIAPTE; Rigano and Strambio-De-Castillia, 2016; 2017) guidelines in combination with the OME-XML data model (Goldberg et al., 2005). In so doing, OMEGA maintains a persistent link between raw image data, intermediate analysis steps, the overall analysis output, and all necessary metadata to repeat the analysis process and reproduce its results.Availability and implementationOMEGA is a cross-platform, open-source software developed in Java. Source code and cross-platform binaries are freely available on GitHub at https://github.com/OmegaProject/Omega (doi: 10.5281/zenodo.2535523), under the GNU General Public License v.3.Contactcaterina.strambio@umassmed.edu and alex.rigano@umassmed.eduSupplementary informationSupplementary Material is available at BioRxiv.org

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“…We next examined M-PMV virion assembly sites at the plasma membrane following the depletion of ROCK1 or LIMK1. To do this, we employed a unique M-PMV Gag-GFP virus developed in the Hunter laboratory (pSARM-GagGFP-M100A) (15). As shown in Fig.…”

Section: Limk1 Plays a Role In Both Hiv-1 And M-pmv Particle Outputmentioning

confidence: 99%

ROCK1 and LIM Kinase Modulate Retrovirus Particle Release and Cell-Cell Transmission Events

Wen

Ding

Wang

et al. 2014

J Virol

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The assembly and release of retroviruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retrovirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retrovirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer monkey virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1-and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retrovirus release from host cells and in cell-cell transmission events. IMPORTANCEViruses often interact with the cellular cytoskeletal machinery in order to deliver their components to the site of assembly and budding. This study indicates that a key regulator of actin dynamics at the plasma membrane, LIM kinase, is important for the release of viral particles for HIV as well as for particle release by a distantly related retrovirus, Mason-Pfizer monkey virus. Moreover, disruption of LIM kinase greatly diminished the spread of HIV from cell to cell. These findings suggest that LIM kinase and its dynamic modulation of the actin cytoskeleton in the cell may be an important host factor for the production, release, and transmission of retroviruses.

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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

Cited by 10 publications

References 32 publications

Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules

Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules

OMEGA: a software tool for the management, analysis, and dissemination of intracellular trafficking data that incorporates motion type classification and quality control

ROCK1 and LIM Kinase Modulate Retrovirus Particle Release and Cell-Cell Transmission Events

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