scholarly journals Visualizing HIV-1 Capsid and Its Interactions with Antivirals and Host Factors

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 246
Author(s):  
Morganne Wilbourne ◽  
Peijun Zhang
Keyword(s):  
Electron Tomography ◽  
Host Factors ◽  
Viral Infectivity ◽  
Hiv Replication ◽  
Nuclear Entry ◽  
Host Chromosome ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
And Function ◽  
Structural Insights

Understanding of the construction and function of the HIV capsid has advanced considerably in the last decade. This is due in large part to the development of more sophisticated structural techniques, particularly cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). The capsid is known to be a pleomorphic fullerene cone comprised of capsid protein monomers arranged into 200–250 hexamers and 12 pentamers. The latter of these induce high curvature necessary to close the cone at both ends. CryoEM/cryoET, NMR, and X-ray crystallography have collectively described these interactions to atomic or near-atomic resolutions. Further, these techniques have helped to clarify the role the HIV capsid plays in several parts of the viral life cycle, from reverse transcription to nuclear entry and integration into the host chromosome. This includes visualizing the capsid bound to host factors. Multiple proteins have been shown to interact with the capsid. Cyclophilin A, nucleoporins, and CPSF6 promote viral infectivity, while MxB and Trim5α diminish the viral infectivity. Finally, structural insights into the intra- and intermolecular interactions that govern capsid function have enabled development of small molecules, peptides, and truncated proteins to disrupt or stabilize the capsid to inhibit HIV replication. The most promising of these, GS6207, is now in clinical trial.

scholarly journals Structural Conservation and Adaptation of the Bacterial Flagella Motor

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1492
Author(s):  
Brittany L. Carroll ◽  
Jun Liu
Keyword(s):  
Electron Tomography ◽  
X Ray ◽  
Bacterial Flagella ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Flagellar Assembly ◽  
Structural Conservation ◽  
And Function ◽  
Structural Insights

Many bacteria require flagella for the ability to move, survive, and cause infection. The flagellum is a complex nanomachine that has evolved to increase the fitness of each bacterium to diverse environments. Over several decades, molecular, biochemical, and structural insights into the flagella have led to a comprehensive understanding of the structure and function of this fascinating nanomachine. Notably, X-ray crystallography, cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) have elucidated the flagella and their components to unprecedented resolution, gleaning insights into their structural conservation and adaptation. In this review, we focus on recent structural studies that have led to a mechanistic understanding of flagellar assembly, function, and evolution.

scholarly journals Structural and mechanistic bases for a potent HIV-1 capsid inhibitor

Science ◽  
2020 ◽  
Vol 370 (6514) ◽  
pp. 360-364 ◽  
Author(s):  
Stephanie M. Bester ◽  
Guochao Wei ◽  
Haiyan Zhao ◽  
Daniel Adu-Ampratwum ◽  
Naseer Iqbal ◽  
...  
Keyword(s):  
Principal Component ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Capsid Shell ◽  
Hydrogen Deuterium ◽  
Infected Cells ◽  
Long Acting ◽  
Structural Insights ◽  
Hiv 1

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo–electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

scholarly journals Meeting Review: 2018 International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 613 ◽  
Author(s):  
Melissa Fernandez ◽  
Eric Freed
Keyword(s):  
International Workshop ◽  
Cytoplasmic Tail ◽  
Structure And Function ◽  
Host Factors ◽  
Host Protein ◽  
Hiv Replication ◽  
Virus Particles ◽  
Recent Developments ◽  
And Function

Recent developments in defining the role of the lentiviral envelope glycoprotein (Env) cytoplasmic tail (CT) in Env trafficking and incorporation into virus particles have advanced our understanding of viral replication and transmission. To stimulate additional progress in this field, the two-day International Workshop on Structure and Function of the Lentiviral gp41 Cytoplasmic Tail, co-organized by Eric Freed and James Hoxie, was held at the National Cancer Institute in Frederick, MD (26–27 April 2018). The meeting served to bring together experts focused on the role of gp41 in HIV replication and to discuss the emerging mechanisms of CT-dependent trafficking, Env conformation and structure, host protein interaction, incorporation, and viral transmission. The conference was organized around the following three main hot topics in gp41 research: the role of host factors in CT-dependent Env incorporation, Env structure, and CT-mediated trafficking and transmission. This review highlights important topics and the advances in gp41 research that were discussed during the conference.

An overview of the recent advances in cryo-electron microscopy for life sciences

2021 ◽  
Author(s):  
Anshul Assaiya ◽  
Ananth Prasad Burada ◽  
Surbhi Dhingra ◽  
Janesh Kumar
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structure Determination ◽  
Electron Tomography ◽  
Protein Structures ◽  
Particle Analysis ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Recent Advances ◽  
Cellular Environment

Cryo-electron microscopy (CryoEM) has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times. It has become indispensable for the characterization of large macromolecular assemblies, membrane proteins, or samples that are limited, conformationally heterogeneous, and recalcitrant to crystallization. Besides, it is the only tool capable of elucidating high-resolution structures of macromolecules and biological assemblies in situ. A state-of-the-art electron microscope operable at cryo-temperature helps preserve high-resolution details of the biological sample. The structures can be determined, either in isolation via single-particle analysis (SPA) or helical reconstruction, electron diffraction (ED) or within the cellular environment via cryo-electron tomography (cryoET). All the three streams of SPA, ED, and cryoET (along with subtomogram averaging) have undergone significant advancements in recent times. This has resulted in breaking the boundaries with respect to both the size of the macromolecules/assemblies whose structures could be determined along with the visualization of atomic details at resolutions unprecedented for cryoEM. In addition, the collection of larger datasets combined with the ability to sort and process multiple conformational states from the same sample are providing the much-needed link between the protein structures and their functions. In overview, these developments are helping scientists decipher the molecular mechanism of critical cellular processes, solve structures of macromolecules that were challenging targets for structure determination until now, propelling forward the fields of biology and biomedicine. Here, we summarize recent advances and key contributions of the three cryo-electron microscopy streams of SPA, ED, and cryoET.

scholarly journals Strain and crack propagation of HIV-1 capsids during uncoating

2021 ◽  
Author(s):  
Alvin Yu ◽  
Elizabeth M.Y Lee ◽  
John A.G. Briggs ◽  
Barbie K Ganser-Pornillos ◽  
Owen Pornillos ◽  
...  
Keyword(s):  
Crack Propagation ◽  
Electron Tomography ◽  
Genetic Material ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Dynamics Simulations ◽  
Capsid Stability ◽  
Hiv 1

Viral replication in HIV-1 relies on a fullerene-shaped capsid to transport genetic material deep into the nucleus of an infected cell. Capsid stability is linked to the presence of cofactors, including inositol hexakisphosphate (IP6) that bind to pores found in the capsid. Using extensive all-atom molecular dynamics simulations of HIV-1 cores imaged from cryo-electron tomography (cryo-ET) in intact virions, which contain IP6 and a ribonucleoprotein complex, we find markedly striated patterns of strain on capsid lattices. The presence of these cofactors also increases rigidity of the capsid. Conformational analysis of capsid (CA) proteins show CA accommodates strain by locally flexing away from structures resolved using x-ray crystallography and cryo-electron microscopy. Then, cryo-ET of HIV-1 cores undergoing endogenous reverse transcription demonstrate that lattice strain increases in the capsid prior to mechanical failure and that the capsid ruptures by crack propagation along regions of high strain. These results uncover HIV-1 capsid properties involved in their critical disassembly process.

IP3 Receptor Plasticity Underlying Diverse Functions

2020 ◽  
Vol 82 (1) ◽  
pp. 151-176 ◽  
Author(s):  
Kozo Hamada ◽  
Katsuhiko Mikoshiba
Keyword(s):  
Structural Changes ◽  
The Body ◽  
Research Progress ◽  
Ip3 Receptor ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
Receptor Plasticity ◽  
Extracellular Stimuli ◽  
And Function

In the body, extracellular stimuli produce inositol 1,4,5-trisphosphate (IP3), an intracellular chemical signal that binds to the IP3 receptor (IP3R) to release calcium ions (Ca2+) from the endoplasmic reticulum. In the past 40 years, the wide-ranging functions mediated by IP3R and its genetic defects causing a variety of disorders have been unveiled. Recent cryo-electron microscopy and X-ray crystallography have resolved IP3R structures and begun to integrate with concurrent functional studies, which can explicate IP3-dependent opening of Ca2+-conducting gates placed ∼90 Å away from IP3-binding sites and its regulation by Ca2+. This review highlights recent research progress on the IP3R structure and function. We also propose how protein plasticity within IP3R, which involves allosteric gating and assembly transformations accompanied by rapid and chronic structural changes, would enable it to regulate diverse functions at cellular microdomains in pathophysiological states.

scholarly journals Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

2021 ◽  
Vol 17 (8) ◽  
pp. e1009775
Author(s):  
Sofia Banchenko ◽  
Ferdinand Krupp ◽  
Christine Gotthold ◽  
Jörg Bürger ◽  
Andrea Graziadei ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Host Restriction ◽  
X Ray Crystallography ◽  
Dynamic Architecture ◽  
Host Restriction Factor ◽  
Differentiated Cells ◽  
Cryo Electron Microscopy ◽  
Ubiquitin Proteasome ◽  
Cycle Regulation ◽  
Structural Insights

Viruses have evolved means to manipulate the host’s ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins.

scholarly journals UVC inactivation of pathogenic samples suitable for cryo-EM analysis

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Jamie S. Depelteau ◽  
Ludovic Renault ◽  
Nynke Althof ◽  
C. Keith Cassidy ◽  
Luiza M. Mendonça ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Electron Tomography ◽  
Test Sample ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Ultraviolet C ◽  
And Function ◽  
Uvc Radiation

AbstractCryo-electron microscopy has become an essential tool to understand structure and function of biological samples. Especially for pathogens, such as disease-causing bacteria and viruses, insights gained by cryo-EM can aid in developing cures. However, due to the biosafety restrictions of pathogens, samples are often treated by chemical fixation to render the pathogen inert, affecting the ultrastructure of the sample. Alternatively, researchers use in vitro or ex vivo models, which are non-pathogenic but lack the complexity of the pathogen of interest. Here we show that ultraviolet-C (UVC) radiation applied at cryogenic temperatures can be used to eliminate or dramatically reduce the infectivity of Vibrio cholerae and the bacterial virus, the ICP1 bacteriophage. We show no discernable structural impact of this treatment of either sample using two cryo-EM methods: cryo-electron tomography followed by sub-tomogram averaging, and single particle analysis (SPA). Additionally, we applied the UVC irradiation to the protein apoferritin (ApoF), which is a widely used test sample for high-resolution SPA studies. The UVC-treated ApoF sample resulted in a 2.1 Å structure indistinguishable from an untreated published map. This research demonstrates that UVC treatment is an effective and inexpensive addition to the cryo-EM sample preparation toolbox.

scholarly journals High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Koji Kato ◽  
Naoyuki Miyazaki ◽  
Tasuku Hamaguchi ◽  
Yoshiki Nakajima ◽  
Fusamichi Akita ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Physiological State ◽  
High Dose ◽  
Final State ◽  
X Ray ◽  
Redox States ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Redox Active ◽  
Beam Damage

AbstractPhotosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

The Cryo-EM Effect: Structural Biology of Neurodegenerative Disease Proteostasis Factors

2021 ◽  
Author(s):  
Benjamin C Creekmore ◽  
Yi-Wei Chang ◽  
Edward B Lee
Keyword(s):  
Neurodegenerative Diseases ◽  
Protein Aggregation ◽  
Neurodegenerative Disease ◽  
Electron Tomography ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
X Ray Crystallography ◽  
New Information ◽  
A Cell ◽  
Regulate Protein

Abstract Neurodegenerative diseases are characterized by the accumulation of misfolded proteins. This protein aggregation suggests that abnormal proteostasis contributes to aging-related neurodegeneration. A better fundamental understanding of proteins that regulate proteostasis may provide insight into the pathophysiology of neurodegenerative disease and may perhaps reveal novel therapeutic opportunities. The 26S proteasome is the key effector of the ubiquitin-proteasome system responsible for degrading polyubiquitinated proteins. However, additional factors, such as valosin-containing protein (VCP/p97/Cdc48) and C9orf72, play a role in regulation and trafficking of substrates through the normal proteostasis systems of a cell. Nonhuman AAA+ ATPases, such as the disaggregase Hsp104, also provide insights into the biochemical processes that regulate protein aggregation. X-ray crystallography and cryo-electron microscopy (cryo-EM) structures not bound to substrate have provided meaningful information about the 26S proteasome, VCP, and Hsp104. However, recent cryo-EM structures bound to substrate have provided new information about the function and mechanism of these proteostasis factors. Cryo-EM and cryo-electron tomography data combined with biochemical data have also increased the understanding of C9orf72 and its role in maintaining proteostasis. These structural insights provide a foundation for understanding proteostasis mechanisms with near-atomic resolution upon which insights can be gleaned regarding the pathophysiology of neurodegenerative diseases.

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