scholarly journals Mechanical Capsid Maturation Facilitates the Resolution of Conflicting Requirements for Herpesvirus Assembly

2021 ◽  
Author(s):  
Alex Evilevitch ◽  
Udom Sae-Ueng
Keyword(s):  
Atomic Force Microscopy ◽  
Viral Particle ◽  
Critical Role ◽  
Dna Packaging ◽  
Maturation Process ◽  
Force Microscopy ◽  
Nano Indentation ◽  
Atomic Force ◽  
Capsid Maturation ◽  
Hsv 1

Most viruses undergo a maturation process from a weakly self-assembled, noninfectious particle to a stable, infectious virion. For herpesviruses, this maturation process resolves several conflicting requirements: i) assembly must be driven by weak, reversible interactions between viral particle subunits to reduce errors and minimize energy of self-assembly; ii) the viral particle must be stable enough to withstand tens of atmospheres of DNA pressure resulting from its strong confinement in the capsid. With herpes simplex virus type 1 (HSV-1) as a prototype of human herpesviruses, we demonstrate that this mechanical capsid maturation is mainly facilitated through capsid-binding auxiliary protein UL25, orthologs of which are present in all herpesviruses. Through genetic manipulation of UL25 mutants of HSV-1 combined with interrogation of capsid mechanics with atomic force microscopy nano-indentation, we suggest the mechanism of stepwise binding of distinct UL25 domains correlated with capsid maturation and DNA packaging. These findings demonstrate another paradigm of viruses as elegantly programmed nano-machines, where an intimate relationship between mechanical and genetic information is preserved in UL25 architecture. IMPORTANCE Minor capsid protein UL25 plays a critical role in mechanical maturation of HSV-1 capsid during virus assembly, required for stable DNA packaging. We modulate UL25-capsid interactions by genetically deleting different UL25 regions and quantify the effect on mechanical capsid stability using an atomic force microscopy (AFM) nano-indentation approach. This approach reveals how UL25 regions reinforce the herpesvirus capsid in order to stably package and retain pressurized DNA. Our data suggests a mechanism of stepwise binding of two main UL25 domains timed with DNA packaging.

UL25 capsid binding facilitates mechanical maturation of the Herpesvirus capsid and allows retention of pressurized DNA

2021 ◽  
Author(s):  
Krista G. Freeman ◽  
Jamie B. Huffman ◽  
Fred L. Homa ◽  
Alex Evilevitch
Keyword(s):  
Dna Packaging ◽  
Maturation Process ◽  
Virion Assembly ◽  
Force Microscopy ◽  
Atomic Force ◽  
Auxiliary Protein ◽  
Capsid Maturation ◽  
Simplex Virus ◽  
Hsv 1

The maturation process that occurs in most viruses is evolutionarily driven as it resolves several conflicting virion assembly requirements. During herpesvirus assembly in a host cell nucleus, micron-long double-stranded herpes DNA is packaged into a nanometer-sized procapsid. This leads to strong confinement of the viral genome with resulting tens of atmospheres of intra-capsid DNA pressure. Yet, the procapsid is unstable due to weak, reversible interactions between its protein subunits, which ensures free energy minimization and reduces assembly errors. In this work we show that herpesviruses resolve these contradictory capsid requirements through a mechanical capsid maturation process facilitated by multi-functional auxiliary protein UL25. Through mechanical interrogation of herpes simplex virus type 1 (HSV-1) capsid with atomic force microscopy nano-indentation, we show that UL25 binding at capsid vertices post-assembly provides the critical capsid reinforcement required for stable DNA encapsidation; the absence of UL25 binding leads to capsid rupture. Furthermore, we demonstrate that gradual capsid reinforcement is a feasible maturation mechanism facilitated by progressive UL25 capsid binding, which is likely correlated with DNA packaging progression. This work provides insight into elegantly programmed viral assembly machinery where targeting of capsid assembly mechanics presents a new antiviral strategy that is resilient to development of drug resistance. Importance: Most viruses undergo a maturation process from a weakly assembled particle to a stable virion. Herpesvirus capsid undergoes mechanical maturation to withstand tens of atmospheres of DNA pressure. We demonstrate that this mechanical capsid maturation is mainly facilitated through binding of auxiliary protein UL25 in HSV-1 capsid vertices. We show that UL25 binding provides the critical capsid reinforcement required for stable DNA encapsidation. Our data also suggests that gradual capsid reinforcement by progressive UL25 binding is a feasible capsid maturation mechanism, correlated with DNA packaging progression.

scholarly journals nanite: using machine learning to assess the quality of atomic force microscopy-enabled nano-indentation data

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Paul Müller ◽  
Shada Abuhattum ◽  
Stephanie Möllmert ◽  
Elke Ulbricht ◽  
Anna V. Taubenberger ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Data Analysis ◽  
Supervised Learning ◽  
Mean Squared Error ◽  
Single Cells ◽  
Contact Point ◽  
Base Line ◽  
Force Microscopy ◽  
Nano Indentation ◽  
Atomic Force

Abstract Background Atomic force microscopy (AFM) allows the mechanical characterization of single cells and live tissue by quantifying force-distance (FD) data in nano-indentation experiments. One of the main problems when dealing with biological tissue is the fact that the measured FD curves can be disturbed. These disturbances are caused, for instance, by passive cell movement, adhesive forces between the AFM probe and the cell, or insufficient attachment of the tissue to the supporting cover slide. In practice, the resulting artifacts are easily spotted by an experimenter who then manually sorts out curves before proceeding with data evaluation. However, this manual sorting step becomes increasingly cumbersome for studies that involve numerous measurements or for quantitative imaging based on FD maps. Results We introduce the Python package nanite, which automates all basic aspects of FD data analysis, including data import, tip-sample separation, base line correction, contact point retrieval, and model fitting. In addition, nanite enables the automation of the sorting step using supervised learning. This learning approach relates subjective ratings to predefined features extracted from FD curves. For ratings ranging from 0 to 10, our approach achieves a mean squared error below 1.0 rating points and a classification accuracy between good and poor curves that is above 87%. We showcase our approach by quantifying Young’s moduli of the zebrafish spinal cord at different classification thresholds and by introducing data quality as a new dimension for quantitative AFM image analysis. Conclusion The addition of quality-based sorting using supervised learning enables a fully automated and reproducible FD data analysis pipeline for biological samples in AFM.

Material Analysis by Ultrasonic Atomic Force Microscopy

2000 ◽  
Vol 627 ◽  
Author(s):  
Chiaki Miyasaka ◽  
Lily Jia ◽  
Bernhard R. Tittmann
Keyword(s):  
Atomic Force Microscopy ◽  
Surface Layer ◽  
Diagnostic Tool ◽  
Ceramic Powders ◽  
Detailed Structure ◽  
Force Microscopy ◽  
Nano Indentation ◽  
Atomic Force ◽  
Good Contrast ◽  
Spray Dried

ABSTRACTSpray-dried ceramic powders (e.g., Al2O3) are composed of a plurality of granules, each of which, includes ceramic particles and organic binders. It is assumed that the binders become concentrated in the surface layer of the granule in accordance with its type or its volume mixed into a ceramic portion of the granule. However, evidence to prove the assumption was limited because conventional microscopes were not able to clearly visualize the segregation. This paper presents a technique for imaging detailed structure of the spray-dried ceramic powders with the ultrasonic-atomic force microscope (U-AFM). The distribution of binder vis-a-vis Al2O3 particles is highly resolved with good contrast. The distribution was confirmed by nano -indentation. Thus, the U-AFM is shown to be a useful diagnostic tool for the development of approaches to spray-dried process evaluation.

Studies of the interphase in epoxy–aluminum joints using nano-indentation and atomic force microscopy

2002 ◽  
Vol 16 (7) ◽  
pp. 935-949 ◽  
Author(s):  
Fuping Li ◽  
John G. Williams ◽  
Burhanettin S. Altan ◽  
Ibrahim Miskioglu ◽  
Robert L. Whipple
Keyword(s):  
Atomic Force Microscopy ◽  
Force Microscopy ◽  
Nano Indentation ◽  
Atomic Force

scholarly journals Mechanical Characterization of Human Corneal Epithelial Cells using Atomic Force Microscopy

2021 ◽  
Author(s):  
Wenyan Yu ◽  
Tong Liu ◽  
Jianqing Mi ◽  
hyock ju Kwon
Keyword(s):  
Mechanical Properties ◽  
Mechanical Property ◽  
Atomic Force Microscopy ◽  
Epithelial Cells ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Force Microscopy ◽  
Nano Indentation ◽  
Atomic Force ◽  
Human Corneal Epithelial Cells

Abstract Background Cell mechanics focuses on the mechanical properties of the cells and how they affect biological behaviors of cells. Mechanical property change of cells can reflect specific diseases and conditions. Therefore, detecting the changes in cell mechanical properties can potentially be associated with diagnosis and treatment of diseases.MethodsMechanical properties of living and dead human corneal epithelial cells (HCECs) were investigated with nano-indentation method using atomic force microscopy (AFM). Sneddon model was adopted to analyze the contacts between pyramidal AFM tip and soft materials. Polyacrylamide (PAAm) substrates with different stiffness were fabricated and human corneal epithelial cells (HCECs) were cultured on them. By applying nano-indentation to HCECs, we examined the distribution of elasticity across cells, and studied the differences between living and dead cells. Findings The central part of a cell, closer to nucleus, was more compliant than the outer regions, toward the surface of the cell’s lipid bilayer. Also, HCECs became stiffer as the stiffness of the substrate increased. Moreover, it was found that dead cells have much higher elastic moduli and exhibited elastic behavior, while living cells showed highly viscoelastic behavior.InterpretationAFM probing is an effective tool for characterizing cell mechanical behavior. More indentation results should be made in further studies to form statistical model of elasticity distribution for HCECs. This study can be extended to investigate the mechanical behaviors of benign and malignant cells or drug interactions on cells, which can establish more probable mechanical property–disease connections.

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