Correlative iPALM and SEM resolves virus cavity and Gag lattice defects in HIV virions

Pedersen, Meike; Jamali, S.H.; Saha, Ipsita; Daum, R.; Bendjennat, Mourad; Saffarian, Saveez

doi:10.1007/s00249-018-1324-0

Cited by 13 publications

(17 citation statements)

References 31 publications

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“…In the same line, the use of Alcian Blue as a solution for grid pre-treatment was shown to increase the adsorption of nanoparticles in this work, thus allowing to improve the detection of these particles avoiding negative staining (Figure 2). Nevertheless, the differentiation of VLPs from EVs was not possible due their similar morphology, but this new label-free method has a potential applicability in combination with X-ray spectroscopy [31], SRFM [53], or "wet" SEM [51] to deepen into nanoparticle characterization. Alternatively, the low contrast present in biological samples can also be overcome in EM by using cryo-TEM, which does not require sample staining (Figure 3).…”

Section: Sample Preparation and Equipment Set-upmentioning

confidence: 99%

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Puente‐Massaguer

Cervera

Gòdia

2020

Viruses

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Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants.

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Section: Sample Preparation and Equipment Set-upmentioning

confidence: 99%

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Puente‐Massaguer

Cervera

Gòdia

2020

Viruses

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“…As mentioned before, the ultra-structural arrangement of the ESCRTs within the neck of the budding vesicles is unknown. It is plausible to use ATP depletion as a tool to lock up the ESCRTs prior to their ultra-structural analysis using either cryotomography [45] or high resolution optical imaging [34,36,46].…”

Section: Plos Onementioning

confidence: 99%

High-speed imaging of ESCRT recruitment and dynamics during HIV virus like particle budding

2020

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Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recruitment in which subunits of ALIX, CHMP4b and VPS4 are recruited with constant proportions on the budding sites of HIV Gag virus like particles for nearly 10 seconds, followed by 2) disassembly of ALIX and CHMP4b while VPS4 signal remains constant for nearly 20 seconds followed by 3) disassembly of VPS4. We hypothesized that the disassembly observed in step 2 was catalyzed by VPS4 and powered by ATP hydrolysis. To test this hypothesis, we performed ATP depletion using (-) glucose medium, deoxyglucose and oligomycin. Imaging ATP depleted cells, we show that the disassembly of CHMP4b and ALIX observed in step 2 is ATP dependent. ATP depletion resulted in the recruitment of approximately 2-fold as many subunits of all ESCRTs. Resuming ATP production in cells, resulted in disassembly of the full ESCRT machinery which had been locked in place during ATP depletion. With some caveats, our experiments provide insight into the formation of the ESCRT machinery at the budding site of HIV during budding.

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“…We have previously imaged purified HIV Gag-Dendra2 VLPs using correlative iPALM and scanning electron microscopy (SEM) and visualized the virion cavity as well as defects within the immature Gag lattice ( 35 ). Although these observations are at a much lower resolution compared to electron cryotomography, iPALM resolves the HIV Gag lattice at ambient temperature within an aqueous environment.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of the HIV Gag Lattice Detected by Localization Correlation Analysis and Time-Lapse iPALM

Saha

Saffarian

2020

Biophysical Journal

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Immature human immunodeficiency virus (HIV) virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. Using electron microscopy, we demonstrate that HIV virus-like particles (VLPs) assembled by the viral protein Gag and tagged at its C-terminus with the fluorescent protein Dendra2 have the same morphology and size as the VLPs assembled using only HIV Gag. We characterize the photophysical properties of Dendra2 and demonstrate that 60% of Dendra2 molecules can be photoswitched and reliably counted in our interferometric photoactivated localization microscopy (iPALM) setup. We further perform iPALM imaging on immobilized HIV Gag-Dendra2 VLPs and demonstrate that we can localize and count 900–1600 Dendra2 molecules within each immobilized VLP with a single-molecule localization precision better than (10 nm) 3 . Our molecular counts correspond to 1400–2400 Gag-Dendra2 proteins incorporated within each VLP. We further calculate temporal correlation functions of localization data, which we present as localization correlation analysis, and show dynamics within the lattice of immobilized VLPs in the timescale of 10–100 s. We further use our localization data to reconstruct time-lapse iPALM images of the Gag-Dendra2 lattice within the lumen of immobilized VLPs. The iPALM time-lapse images show significant lattice dynamics within the lumen of VLPs. Addition of disuccinimidyl suberate to the VLPs completely abrogated these dynamics as observed in both localization correlation analysis and time-lapse iPALM. In a complementary approach, we utilized HaXS8 cross-linking reactions between Halo and SNAP proteins and verified lattice dynamics in purified VLPs incorporating 10% Gag-SNAP, 10% Gag-Halo, and 80% Gag proteins. The HIV Gag lattice, along with the structural lattice of other enveloped viruses, has been mostly considered static. Our study provides an important tool to investigate the dynamics within these enveloped viruses.

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Correlative iPALM and SEM resolves virus cavity and Gag lattice defects in HIV virions

Cited by 13 publications

References 31 publications

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

High-speed imaging of ESCRT recruitment and dynamics during HIV virus like particle budding

Dynamics of the HIV Gag Lattice Detected by Localization Correlation Analysis and Time-Lapse iPALM

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