scholarly journals Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

2016 ◽  
Vol 90 (19) ◽  
pp. 8542-8551 ◽  
Author(s):  
Lauren M. Drouin ◽  
Bridget Lins ◽  
Maria Janssen ◽  
Antonette Bennett ◽  
Paul Chipman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Intramolecular Interactions ◽  
Single Amino Acid ◽  
Fold Axis ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Genome Packaging ◽  
Cryo Electron Microscopy ◽  
Empty Capsid ◽  
Packaging Efficiency

ABSTRACTThe adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.IMPORTANCEThe mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

scholarly journals Structural and functional analysis of protective antibodies targeting the threefold plateau of enterovirus 71

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kuan-Ying A. Huang ◽  
Daming Zhou ◽  
Elizabeth E. Fry ◽  
Abhay Kotecha ◽  
Peng-Nien Huang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neutralizing Antibodies ◽  
Enterovirus 71 ◽  
Therapeutic Agents ◽  
Fold Axis ◽  
Effective Protection ◽  
Promising Candidate ◽  
Cryo Electron Microscopy ◽  
Protective Antibodies ◽  
Conserved Epitope

Abstract Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.

scholarly journals Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

2009 ◽  
Vol 391 (2) ◽  
pp. 471-483 ◽  
Author(s):  
Robert L. Duda ◽  
Philip D. Ross ◽  
Naiqian Cheng ◽  
Brian A. Firek ◽  
Roger W. Hendrix ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Calorimetry ◽  
Cryo Electron Microscopy

Cryo-Electron Microscopy of Aura Viruses

1998 ◽  
Vol 4 (S2) ◽  
pp. 946-947
Author(s):  
W. Zhang ◽  
N. H. Olson ◽  
B. R. McKinney ◽  
R. J. Kuhn ◽  
T. S. Baker
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Single Copy ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Subgenomic Rna ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Cryo Electron Microscopy

Alphaviruses are a group of enveloped viruses in the Togaviridae family. Studies of several alphaviruses, including Ross River, Sindbis and Semliki Forest viruses, by cryo-electron microscopy (cryo-EM), three-dimensional (3D) image resconstruction and other techniques have illustrated that these spherical viruses have a T=4, multi-layered structure.Aura virus, which is closely related to Sindbis, was first isolated in South America. Unlike the other alphaviruses, both genomic RNA (12kb, 49S) and subgenomic RNA(4.2kb, 26S) are encapsidated efficiently and form mature virions. Studies on negatively-stained virus particles demonstrated that there are two major size classes. The first contains particles of ∼72nm diameter, which are most similar to wild type virus, whereas the second class includes particles of ∼62nm in diameter. The 72nm particles are believed to have one copy of genomic RNA or one to three copies of subgenomic RNA, and a T=4 structure. The 62nm particles probably only have a single copy of subgenomic RNA and are presumed to be T=3 structures.

scholarly journals Influenza A virus defective viral genomes are inefficiently packaged into virions relative to wild-type genomic RNAs

2021 ◽  
Author(s):  
Fadi G Alnaji ◽  
William K Reiser ◽  
Aartjan te Velthuis ◽  
Christopher B Brooke
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Wild Type ◽  
Genome Packaging ◽  
Genomic Rnas ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Packaging Efficiency

Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hotspots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild type genomic RNAs. Surprisingly, packaged DelVGs exhibited no signs of enrichment for specific deletion characteristics suggesting that all DelVGs are equally limited in packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the IAV replication machinery and shed light on the mechanisms that underly DelVG formation.

scholarly journals Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

2022 ◽  
Vol 119 (2) ◽  
pp. e2114994119
Author(s):  
Benjamin J. LaFrance ◽  
Johanna Roostalu ◽  
Gil Henkin ◽  
Basil J. Greber ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Total Internal Reflection ◽  
Dynamic Instability ◽  
Wild Type ◽  
Gtp Hydrolysis ◽  
Cryo Electron Microscopy ◽  
Catalytically Active ◽  
The Stability ◽  
Insight Into

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

scholarly journals Structural Dynamics of Nonenveloped Virus Disassembly Intermediates

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Kimi Azad ◽  
Manidipa Banerjee
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Icosahedral Symmetry ◽  
Fold Axis ◽  
Three Dimensional Reconstruction ◽  
Cryo Electron Microscopy ◽  
Successful Infection ◽  
Dimensional Reconstruction ◽  
Nonenveloped Virus

ABSTRACT The stability of icosahedral viruses is crucial for protecting the viral genome during transit; however, successful infection requires eventual disassembly of the capsid. A comprehensive understanding of how stable, uniform icosahedrons disassemble remains elusive, mainly due to the complexities involved in isolating transient intermediates. We utilized incremental heating to systematically characterize the disassembly pathway of a model nonenveloped virus and identified an intriguing link between virus maturation and disassembly. Further, we isolated and characterized two intermediates by cryo-electron microscopy and three-dimensional reconstruction, without imposing icosahedral symmetry. The first intermediate displayed a series of major, asymmetric alterations, whereas the second showed that the act of genome release, through the 2-fold axis, is actually confined to a small section on the capsid. Our study thus presents a comprehensive structural analysis of nonenveloped virus disassembly and emphasizes the asymmetric nature of programmed conformational changes. IMPORTANCE Disassembly or uncoating of an icosahedral capsid is a crucial step during infection by nonenveloped viruses. However, the dynamic and transient nature of the disassembly process makes it challenging to isolate intermediates in a temporal, stepwise manner for structural characterization. Using controlled, incremental heating, we isolated two disassembly intermediates: “eluted particles” and “puffed particles” of an insect nodavirus, Flock House virus (FHV). Cryo-electron microscopy and three-dimensional reconstruction of the FHV disassembly intermediates indicated that disassembly-related conformational alterations are minimally global and largely local, leading to asymmetry in the particle and eventual genome release without complete disintegration of the icosahedron.

scholarly journals Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants

2021 ◽  
Author(s):  
James W. Saville ◽  
Dhiraj Mannar ◽  
Xing Zhu ◽  
Shanti S. Srivastava ◽  
Alison M. Berezuk ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Viral Fitness ◽  
Spike Protein ◽  
Wild Type ◽  
Antibody Neutralization ◽  
Vaccination Rates ◽  
Modest Increase ◽  
Terminal Domain ◽  
Cryo Electron Microscopy ◽  
Neutralizing Epitopes

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

scholarly journals ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Charlotte Lässig ◽  
Sarah Matheisl ◽  
Konstantin MJ Sparrer ◽  
Carina C de Oliveira Mann ◽  
Manuela Moldt ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Innate Immune ◽  
Viral Rna ◽  
Wild Type ◽  
Cryo Electron Microscopy ◽  
Biochemical Studies ◽  
Infected Cells ◽  
System Disorder

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.

scholarly journals Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

2014 ◽  
Vol 89 (3) ◽  
pp. 1794-1808 ◽  
Author(s):  
Yu-Shan Tseng ◽  
Brittney L. Gurda ◽  
Paul Chipman ◽  
Robert McKenna ◽  
Sandra Afione ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Clinical Trials ◽  
Image Reconstruction ◽  
Gene Delivery ◽  
Delivery System ◽  
Fold Axis ◽  
Adeno Associated Virus ◽  
Cryo Electron Microscopy ◽  
Antigenic Sites ◽  
Serotype 1

ABSTRACTThe clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions.IMPORTANCEThe adeno-associated viruses (AAVs) are promising vectors forin vivotherapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that report therapeutic efficacy. However, a large percentage of the population has preexisting AAV capsid antibodies and therefore must be excluded from clinical trials or vector readministration. This report represents our continuing efforts to understand the antigenic structure of the AAVs, specifically, to obtain a picture of “polyclonal” reactivity as is the situation in humans. It describes the structures of four AAV-antibody complexes determined by cryo-electron microscopy and image reconstruction, increasing the number of mapped epitopes to four and three, respectively, for AAV1 and AAV5, two vectors currently in clinical trials. The results presented provide information essential for generating antigenic escape vectors to overcome a critical challenge remaining in the optimization of this highly promising vector delivery system.

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