Review

Authors: Aditya Raguram, V Sasisekharan, Ram Sasisekharan...

Recent developments of rational strategies for the design of antiviral therapies, including monoclonal antibodies (mAbs), have naturally relied extensively on available viral structural information. As new strategies continue to be developed, it is equally important to continue to refine our understanding and interpretation of viral structural data. There are known limitations to the traditional (Caspar-Klug) theory for describing virus capsid structures that involves subdividing a capsid into triangular subunits. In this context, we describe a more general polyhedral framework for describing virus capsid structures that is able to account for many of these limitations, including a more thorough characterization of intersubunit interfaces. Additionally, our use of pentagonal subunits instead of triangular ones accounts for the intrinsic chirality observed in all capsids. In conjunction with the existing theory, the framework presented here provides a more complete picture of a capsid's structure and therefore can help contribute to the development of more effective antiviral strategies.

Topics: Capsid; Capsid Proteins; Models, Biological; Protein Conformation; Viral Structural Proteins; Virus Assembly

PubMed: 28094093
DOI: 10.1016/j.tim.2016.12.007

Authors: Mario Mietzsch, Courtnee Eddington, Ariana Jose...

Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new , genes resulting in hundreds of natural and engineered AAV capsid variants, while the gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 was replaced with the gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2- system. In further experiments the 3' end of the AAV2 gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. A major by-product of all adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Topics: Capsid; Capsid Proteins; DNA-Binding Proteins; Dependovirus; Genes, Viral; Genetic Vectors; HEK293 Cells; Humans; Recombinant Fusion Proteins; Viral Genome Packaging; Viral Proteins

PubMed: 34287038
DOI: 10.1128/JVI.00773-21

Review

Authors: Joy Twentyman, Michael Emerman, Molly Ohainle...

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as through , have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

Topics: Animals; Capsid; Capsid Proteins; Lentivirus; Host-Pathogen Interactions; Lentivirus Infections

PubMed: 38497663
DOI: 10.1128/jvi.00308-24

Authors: Caryn L Heldt, Oluwatoyin Areo, Pratik U Joshi...

Adeno-associated virus (AAV) is showing promise as a therapy for diseases that contain a single-gene deletion or mutation. One major scale-up challenge is the removal of empty or non-gene of interest containing AAV capsids. Analytically, the empty capsids can be separated from full capsids using anion exchange chromatography. However, when scaled up to manufacturing, the minute changes in conductivity are difficult to consistently obtain. To better understand the differences in the empty and full AAV capsids, we have developed a single-particle atomic force microscopy (AFM) method to measure the differences in the charge and hydrophobicity of AAV capsids at the single-particle level. The atomic force microscope tip was functionalized with either a charged or a hydrophobic molecule, and the adhesion force between the functionalized atomic force microscope tip and the virus was measured. We measured a change in the charge and hydrophobicity between empty and full AAV2 and AAV8 capsids. The charge and hydrophobicity differences between AAV2 and AAV8 are related to the distribution of charge on the surface and not the total charge. We propose that the presence of nucleic acids inside the capsid causes minor but measurable changes in the capsid structure that lead to measurable surface changes in charge and hydrophobicity.

Topics: Capsid; Dependovirus; Microscopy, Atomic Force; Capsid Proteins; Genetic Vectors

PubMed: 37040364
DOI: 10.1021/acs.langmuir.2c02643

Review

Authors: Estrella Lopez-Gordo, Kyle Chamberlain, Jalish Mahmud Riyad...

Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in molecular engineering, high-throughput screening platforms, and computational techniques have resulted in a toolbox of capsid variants with enhanced performance over parental serotypes. Despite their considerable promise and emerging clinical success, there are still obstacles hindering their broader use, including limited transduction capabilities, tissue/cell type-specific tropism and penetration into tissues through anatomical barriers, off-target tissue biodistribution, intracellular degradation, immune recognition, and a lack of translatability from preclinical models to clinical settings. Here, we first describe the transduction mechanisms of natural AAV serotypes and explore the current understanding of the systemic and cellular hurdles to efficient transduction. We then outline progress in developing designer AAV capsid variants, highlighting the seminal discoveries of variants which can transduce the central nervous system upon systemic administration, and, to a lesser extent, discuss the targeting of the peripheral nervous system, eye, ear, lung, liver, heart, and skeletal muscle, emphasizing their tissue and cell specificity and translational promise. In particular, we dive deeper into the molecular mechanisms behind their enhanced properties, with a focus on their engagement with host cell receptors previously inaccessible to natural AAV serotypes. Finally, we summarize the main findings of our review and discuss future directions.

Topics: Capsid; Dependovirus; Serogroup; Tissue Distribution; Capsid Proteins; Tropism; Genetic Vectors

PubMed: 38543807
DOI: 10.3390/v16030442

Review

Authors: Wenhan Guo, Esther Alarcon, Jason E Sanchez...

Viral capsid assembly is a complex and critical process, essential for understanding viral behavior, evolution, and the development of antiviral treatments, vaccines, and nanotechnology. Significant progress in studying viral capsid assembly has been achieved through various computational approaches, including molecular dynamics (MD) simulations, stochastic dynamics simulations, coarse-grained (CG) models, electrostatic analyses, lattice models, hybrid techniques, machine learning methods, and kinetic models. Each of these techniques offers unique advantages, and by integrating these diverse computational strategies, researchers can more accurately model the dynamic behaviors and structural features of viral capsids, deepening our understanding of the assembly process. This review provides a comprehensive overview of studies on viral capsid assembly, emphasizing their critical role in advancing our knowledge. It examines the contributions, strengths, and limitations of different computational methods, presents key computational works in the field, and analyzes milestone studies that have shaped current research.

Topics: Capsid; Virus Assembly; Molecular Dynamics Simulation; Capsid Proteins; Humans

PubMed: 39768179
DOI: 10.3390/cells13242088

Authors: Iris Seitz, Sharon Saarinen, Esa-Pekka Kumpula...

Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. Importantly, precise control over the size and shape of virus capsids would have advantages in the development of new vaccines and delivery systems. However, current tools to direct the assembly process in a programmable manner are exceedingly elusive. Here we introduce a modular approach by demonstrating DNA-origami-directed polymorphism of single-protein subunit capsids. We achieve control over the capsid shape, size and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms, which are efficiently encapsulated within the capsid. Furthermore, the obtained viral capsid coatings can shield the encapsulated DNA origami from degradation. Our approach is, moreover, not limited to a single type of capsomers and can also be applied to RNA-DNA origami structures to pave way for next-generation cargo protection and targeting strategies.

Topics: Capsid; Capsid Proteins; Nanostructures; DNA; Virion

PubMed: 37460794
DOI: 10.1038/s41565-023-01443-x

Authors: Qin Huang, Albert T Chen, Ken Y Chan...

Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood-brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.

Topics: Mice; Animals; Blood-Brain Barrier; Capsid; Genetic Vectors; Central Nervous System; Capsid Proteins; Dependovirus

PubMed: 37467291
DOI: 10.1371/journal.pbio.3002112

Review

Authors: Glen R Nemerow, Phoebe L Stewart, Vijay S Reddy...

A detailed structural analysis of the entire human adenovirus capsid has been stymied by the complexity and size of this 150 MDa macromolecular complex. Over the past 10 years, the steady improvements in viral genome manipulation concomitant with advances in crystallographic techniques and data processing software has allowed structure determination of this virus by X-ray diffraction at 3.5 Å resolution. The virus structure revealed the location, folds, and interactions of major and minor (cement proteins) on the inner and outer capsid surface. This new structural information sheds further light on the process of adenovirus capsid assembly and virus-host cell interactions.

Topics: Adenovirus Infections, Human; Adenoviruses, Human; Animals; Capsid; Capsid Proteins; Humans; X-Ray Diffraction

PubMed: 22482707
DOI: 10.1016/j.coviro.2011.12.008

Authors: Manutea C Serrero, Virginie Girault, Sebastian Weigang...

Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid-host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.

Topics: Capsid; Capsid Proteins; GTP Phosphohydrolases; Herpesviridae; Interferons; Simplexvirus

PubMed: 35475759
DOI: 10.7554/eLife.76804