Monitoring HIV-1 Assembly in Living Cells: Insights from Dynamic and Single Molecule Microscopy

Inamdar, Kaushik; Floderer, Charlotte; Favard, Cyril; Muriaux, Delphine

doi:10.3390/v11010072

Cited by 34 publications

(23 citation statements)

References 78 publications

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“…The PSD of VLPs has been studied with the six methods evaluated in this work. Typically, the VLP mean diameter is comprised between 100 and 200 nm, as observed by TEM, cryo-TEM, NTA and flow virometry [61]. Non-symmetric right-skewed distributions were observed for VLPs measured with the different techniques.…”

Section: Particle Size Distributionmentioning

confidence: 92%

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: Particle Size Distributionmentioning

confidence: 92%

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

Puente‐Massaguer

Cervera

Gòdia

2020

Viruses

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“…These findings provide insights into assembly of the immature virus, its structure, and how it disassembles during maturation. still unclear whether these are Gag dimers, trimers, or other multimeric forms of Gag (16,17).…”

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confidence: 99%

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation

Tan

Pak

Morado

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intrahexameric and interhexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the oligomerization state of CA at lattice edges and found that CA forms partial hexamers. These structures reveal the network of interactions formed by CA-SP1 at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, partial hexamers exist at lattice edges, these are in a helix-coil dynamic equilibrium, and partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation.

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“…In this regard, recent improvements in confocal microscopy have enabled the study of the assembly process of different viruses (19). Live cell characterization up to single-molecule level has been accomplished by the combination of high-speed acquisition and advanced super-resolution microscopy techniques (20,21). In parallel, cryogenic transmission electron microscopy (cryo-TEM) has emerged as a powerful tool to evaluate the structure and morphology of nanoparticles (22,23).…”

mentioning

confidence: 99%

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

et al. 2020

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Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag‐based virus‐like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo‐TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion‐derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23‐fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag‐eGFP polyprotein were obtained in High Five cells (3.6‐fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7‐ and 1.5‐fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five‐derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry

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Monitoring HIV-1 Assembly in Living Cells: Insights from Dynamic and Single Molecule Microscopy

Cited by 34 publications

References 78 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

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

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