Detection and Tracking of Dual-Labeled HIV Particles Using Wide-Field Live Cell Imaging to Follow Viral Core Integrity

Mamede, João I.; Hope, Thomas J.

doi:10.1007/978-1-4939-3046-3_4

Cited by 13 publications

(13 citation statements)

References 15 publications

(18 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Second, we assessed the role of fusion using HIV-1 virions incorporating betalactamase-Vpr (BlaM-Vpr). In this system, fusion can be detected following loading of cells with CCF2 as this fluorescent dye is a target of BlaM and, following its cleavage, its fluorescence emission changes to the blue spectrum (447 nm) 44,45 . Importantly, CCF2 cleavage was not altered by nutrient deprivation nor by α-KG supplementation, indicating that these metabolic changes modulated infection at a post fusion level ( Supplementary Fig.…”

Section: Inhibition Of Aerobic Glycolysis Increases Hiv-1 Infectionmentioning

confidence: 99%

Entry of glucose- and glutamine-derived carbons into the citric acid cycle supports early steps of HIV-1 infection in CD4 T cells

et al. 2019

Self Cite

View full text Add to dashboard Cite

The susceptibility of CD4 T cells to human immunodeficiency virus 1 (HIV-1) infection is regulated by glucose and glutamine metabolism, but the relative contributions of these nutrients to infection are not known. Here we show that glutaminolysis is the major pathway fuelling the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in T-cell receptorstimulated naïve, as well as memory CD4, subsets and is required for optimal HIV-1 infection. Under conditions of attenuated glutaminolysis, the α-ketoglutarate (α-KG) TCA rescues early steps in infection; exogenous α-KG promotes HIV-1 reverse transcription, rendering both naïve and memory cells more sensitive to infection. Blocking the glycolytic flux of pyruvate to lactate results in altered glucose carbon allocation to TCA and pentose phosphate pathway intermediates, an increase in OXPHOS and augmented HIV-1 reverse transcription. Moreover, HIV-1 infection is Reprints and permissions information is available at www.nature.com/reprints.

show abstract

Section: Inhibition Of Aerobic Glycolysis Increases Hiv-1 Infectionmentioning

confidence: 99%

Entry of glucose- and glutamine-derived carbons into the citric acid cycle supports early steps of HIV-1 infection in CD4 T cells

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, many of the fundamental image data management limitations holding back "[…] the routine application of automated image analysis […] to large volumes of information generated by digital imaging" (verbatim from: Swedlow et al, 2003) are still in place even several years after the initial identification of the problem. As a case in point, the utilization of viral particle tracking to draw direct real-time correlations between alterations in viral mobility and underlying perturbations in the viral and cellular states, remains a considerable challenge even at low-throughput, and it is difficult if not impossible to scale to the systems biology level (Arhel et al, 2006;McDonald et al, 2002;Mamede et al, 2017;Mamede and Hope, 2016;Sood et al, 2017). As a result, most virology studies to date rely on biochemical and genetic analyses conducted in bulk and on the microscopic analysis of fixed cells, which fail to capture viral heterogeneity and the complexity of viral infection processes.…”

Section: Description Of the Problemmentioning

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

View full text Add to dashboard Cite

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

show abstract

“…Even point mutations in CA can compromise infectivity through altering capsid assembly and/or stability (e.g., [ 5 ]). A recently introduced microscopy assay that may indirectly monitor HIV-1 uncoating live cells is based upon the assertion that a fraction of GFP molecules produced upon cleavage of Gag-iGFP precursor [ 13 ] is trapped within an intact mature core and is released at the time of uncoating [ 14 , 15 ]. However, further validation of the notion that a detectable number of GFP molecules is trapped in a considerable fraction of mature cores is needed.…”

Section: Introductionmentioning

confidence: 99%

Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells

et al. 2016

View full text Add to dashboard Cite

Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process.

show abstract

Detection and Tracking of Dual-Labeled HIV Particles Using Wide-Field Live Cell Imaging to Follow Viral Core Integrity

Cited by 13 publications

References 15 publications

Entry of glucose- and glutamine-derived carbons into the citric acid cycle supports early steps of HIV-1 infection in CD4 T cells

Entry of glucose- and glutamine-derived carbons into the citric acid cycle supports early steps of HIV-1 infection in CD4 T cells

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

Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells

Contact Info

Product

Resources

About