scholarly journals Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

2021 ◽  
Author(s):  
Shimon Harel ◽  
Yarin Altaras ◽  
Dikla Nachmias ◽  
Noa Rotem-Dai ◽  
Inbar Segal ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Cell Lines ◽  
Particle Analysis ◽  
Fast Kinetics ◽  
Bud Formation ◽  
Force Microscopy ◽  
Virion Release ◽  
Atomic Force ◽  
Vacuolar Protein ◽  
Hiv 1

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Whereas the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (< 100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wildtype (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 30% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

scholarly journals PF74 Reinforces the HIV-1 Capsid To Impair Reverse Transcription-Induced Uncoating

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Sanela Rankovic ◽  
Ruben Ramalho ◽  
Christopher Aiken ◽  
Itay Rousso
Keyword(s):  
Atomic Force Microscopy ◽  
Small Molecule ◽  
Reverse Transcription ◽  
Direct Evidence ◽  
Main Body ◽  
Force Microscopy ◽  
The Core ◽  
Atomic Force ◽  
Capsid Stability ◽  
Hiv 1

ABSTRACTThe RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here, we show, using atomic force microscopy (AFM), that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilizes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 μM, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure that was observed to form during reverse transcriptionin vitropersisted throughout the duration of the measurement (∼24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription while interfering with a late step in uncoating.IMPORTANCEThe capsid-binding small molecule PF74 inhibits HIV-1 infection at early stages and perturbs uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. We recently introduced time-lapse atomic force microscopy to study the morphology and physical properties of HIV-1 cores during the course of reverse transcription. Here, we apply this AFM methodology to show that PF74 prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, but a cap-like structure dissociated from the narrow end of the core HIV-1. Our result provides direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice.

scholarly journals Nucleoprotein Intermediates in HIV-1 DNA Integration Visualized by Atomic Force Microscopy

2010 ◽  
Vol 399 (3) ◽  
pp. 491-500 ◽  
Author(s):  
Svetlana Kotova ◽  
Min Li ◽  
Emilios K. Dimitriadis ◽  
Robert Craigie
Keyword(s):  
Atomic Force Microscopy ◽  
Dna Integration ◽  
Force Microscopy ◽  
Atomic Force ◽  
Hiv 1

scholarly journals Identification of HIV-1–Based Virus-like Particles by Multifrequency Atomic Force Microscopy

2016 ◽  
Vol 111 (6) ◽  
pp. 1173-1179 ◽  
Author(s):  
Irene González-Domínguez ◽  
Sonia Gutiérrez-Granados ◽  
Laura Cervera ◽  
Francesc Gòdia ◽  
Neus Domingo
Keyword(s):  
Atomic Force Microscopy ◽  
Virus Like Particles ◽  
Force Microscopy ◽  
Atomic Force ◽  
Hiv 1

scholarly journals Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines

Open Biology ◽  
2014 ◽  
Vol 4 (5) ◽  
pp. 140046 ◽  
Author(s):  
Jan Rother ◽  
Helen Nöding ◽  
Ingo Mey ◽  
Andreas Janshoff
Keyword(s):  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Cell Lines ◽  
Loss Tangent ◽  
Structural Changes ◽  
Metastatic Potential ◽  
Adhesive Properties ◽  
Force Microscopy ◽  
Atomic Force ◽  
Independent Information

Mechanical phenotyping of cells by atomic force microscopy (AFM) was proposed as a novel tool in cancer cell research as cancer cells undergo massive structural changes, comprising remodelling of the cytoskeleton and changes of their adhesive properties. In this work, we focused on the mechanical properties of human breast cell lines with different metastatic potential by AFM-based microrheology experiments. Using this technique, we are not only able to quantify the mechanical properties of living cells in the context of malignancy, but we also obtain a descriptor, namely the loss tangent, which provides model-independent information about the metastatic potential of the cell line. Including also other cell lines from different organs shows that the loss tangent ( G″ / G′ ) increases generally with the metastatic potential from MCF-10A representing benign cells to highly malignant MDA-MB-231 cells.

scholarly journals Time- and Zinc-Related Changes in Biomechanical Properties of Human Colorectal Cancer Cells Examined by Atomic Force Microscopy

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 468
Author(s):  
Maria Maares ◽  
Claudia Keil ◽  
Leif Löher ◽  
Andreas Weber ◽  
Amsatou Andorfer-Sarr ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Cell Lines ◽  
Mechanical Response ◽  
Cultured Cells ◽  
Force Microscopy ◽  
Atomic Force ◽  
The Impact ◽  
Ht 29

Monitoring biomechanics of cells or tissue biopsies employing atomic force microscopy (AFM) offers great potential to identify diagnostic biomarkers for diseases, such as colorectal cancer (CRC). Data on the mechanical properties of CRC cells, however, are still scarce. There is strong evidence that the individual zinc status is related to CRC risk. Thus, this study investigates the impact of differing zinc supply on the mechanical response of the in vitro CRC cell lines HT-29 and HT-29-MTX during their early proliferation (24–96 h) by measuring elastic modulus, relaxation behavior, and adhesion factors using AFM. The differing zinc supply severely altered the proliferation of these cells and markedly affected their mechanical properties. Accordingly, zinc deficiency led to softer cells, quantitatively described by 20–30% lower Young’s modulus, which was also reflected by relevant changes in adhesion and rupture event distribution compared to those measured for the respective zinc-adequate cultured cells. These results demonstrate that the nutritional zinc supply severely affects the nanomechanical response of CRC cell lines and highlights the relevance of monitoring the zinc content of cancerous cells or biopsies when studying their biomechanics with AFM in the future.

Analysis of environmental particles by atomic force microscopy, scanning and transmission electron microscopy

2004 ◽  
Vol 50 (12) ◽  
pp. 9-18 ◽  
Author(s):  
D. Mavrocordatos ◽  
W. Pronk ◽  
M. Boller
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Nano Particles ◽  
Particle Analysis ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transmission Electron ◽  
The Individual ◽  
Amorphous Particles

Due to their large specific surface and their abundance, micro and nano particles play an important role in the transport of micropollutants in the environment. Natural particles are usually composed of a mixture of inorganic amorphous or crystalline material (mainly FeOOH, FexOy, MnxOy and clays) and organic material (humics and polysaccharides). They all tend to occur as very small particles (1-1,000 nm in diameter). Most natural amorphous particles are unstable and tend to transform with time towards more crystalline forms, either by aging or possibly, by dissolution and re-crystallization. Such transformations affect the fate of sorbed micropollutants and the scavenging properties are therefore changed. As these entities are sensitive to dehydration (aggregation, changes in the morphology), it is highly important to observe their morphology in their natural environment and understand their composition at the scale of the individual particles. Also for the understanding and optimization of water treatment technologies, the knowledge of the occurrence and behavior of nano-particles is of high importance. Some of the possible particle analysis methods are presented: aggregation processes, biomineralization, bacterial adhesion, biofilms in freshwaters, ferrihydrite as heavy metals remover from storm water. These examples demonstrate the capabilities and focus of the microscopes. Atomic Force Microscopy (AFM) allows to analyze the particles in their own environment, meaning in air or in the water. Thus, native aspects of particles can be observed. As well, forces of interactions between particles or between particles and other surfaces such as membranes will be highly valuable data. Scanning Electron Microscopy (SEM) and for higher lateral resolution, Transmission Electron Microscopy (TEM) allow measurement of the morphology and composition. Especially, TEM coupled with Electron Energy Loss Spectroscopy (TEM-EELS) is a powerful technique for elemental analysis. Finally, general guidelines for the effective use of microscopic techniques are provided.

scholarly journals Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy

PLoS ONE ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. e0228036
Author(s):  
Shaolong Chen ◽  
Jun Xu ◽  
Mingyue Liu ◽  
A. L. N. Rao ◽  
Roya Zandi ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Force Microscopy ◽  
Atomic Force ◽  
Hiv 1
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