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Langmuir : the ACS Journal of Surfaces... Apr 2023Adeno-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...
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 -
Journal of Virology Sep 2021Recombinant 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...
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 -
Current Opinion in Virology Jun 2019During retrovirus maturation, cleavage of the precursor structural Gag polyprotein by the viral protease induces architectural rearrangement of the virus particle from... (Review)
Review
During retrovirus maturation, cleavage of the precursor structural Gag polyprotein by the viral protease induces architectural rearrangement of the virus particle from an immature into a mature, infectious form. The structural rearrangement encapsidates the viral RNA genome in a fullerene capsid, producing a diffusible viral core that can initiate infection upon entry into the cytoplasm of a host cell. Maturation is an important therapeutic window against HIV-1. In this review, we highlight recent breakthroughs in understanding of the structures of retroviral immature and mature capsid lattices that define the boundary conditions of maturation and provide novel insights on capsid transformation. We also discuss emerging insights on encapsidation of the viral genome in the mature capsid, as well as remaining questions for further study.
Topics: Capsid; Capsid Proteins; Genome, Viral; HIV-1; Models, Molecular; Peptide Hydrolases; RNA, Viral; Retroviridae; Virion; Virus Assembly
PubMed: 31185449
DOI: 10.1016/j.coviro.2019.05.004 -
Trends in Microbiology Jun 2017Recent developments of rational strategies for the design of antiviral therapies, including monoclonal antibodies (mAbs), have naturally relied extensively on available... (Review)
Review
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 -
Journal of Virology Apr 2024Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the... (Review)
Review
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 -
Viruses Jan 2017Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a... (Review)
Review
Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a capsid protein shell and a positive-sense, single-stranded RNA genome. The capsid protein undergoes dramatic proteolytic processing both inside and outside of the host cell, resulting in a coordinated maturation process that affects cellular localization, virus structure, and infectivity. After maturation, the capsid protein controls the initial phases of virus infection, including virus attachment, endocytosis, and genome release into the host cell. The astrovirus capsid is the target of host antibodies including virus-neutralizing antibodies. The capsid protein also mediates the binding of host complement proteins and inhibits complement activation. Here, we will review our knowledge on the astrovirus capsid protein (CP), with particular attention to the recent structural, biochemical, and virological studies that have advanced our understanding of the astrovirus life cycle.
Topics: Animals; Astroviridae Infections; Capsid; Capsid Proteins; Humans; Mamastrovirus
PubMed: 28106836
DOI: 10.3390/v9010015 -
Nature Nanotechnology Oct 2023Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. Importantly, precise control over the size and shape of...
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 -
Current Opinion in Virology Apr 2012A 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... (Review)
Review
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 -
PLoS Pathogens Aug 2022In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following...
In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.
Topics: Humans; Active Transport, Cell Nucleus; Capsid; Capsid Proteins; HIV Infections; HIV-1; Virion; Virus Replication; Virus Uncoating; Virus Integration
PubMed: 35951676
DOI: 10.1371/journal.ppat.1010754 -
Nature Microbiology Oct 2020As the first discovered human cancer virus, Epstein-Barr virus (EBV) causes Burkitt's lymphoma and nasopharyngeal carcinoma. Isolating virions for determining...
As the first discovered human cancer virus, Epstein-Barr virus (EBV) causes Burkitt's lymphoma and nasopharyngeal carcinoma. Isolating virions for determining high-resolution structures has been hindered by latency-a hallmark of EBV infection-and atomic structures are thus available only for recombinantly expressed EBV proteins. In the present study, by symmetry relaxation and subparticle reconstruction, we have determined near-atomic-resolution structures of the EBV capsid with an asymmetrically attached DNA-translocating portal and capsid-associated tegument complexes from cryogenic electron microscopy images of just 2,048 EBV virions obtained by chemical induction. The resulting atomic models reveal structural plasticity among the 20 conformers of the major capsid protein, 2 conformers of the small capsid protein (SCP), 4 conformers of the triplex monomer proteins and 2 conformers of the triplex dimer proteins. Plasticity reaches the greatest level at the capsid-tegument interfaces involving SCP and capsid-associated tegument complexes (CATC): SCPs crown pentons/hexons and mediate tegument protein binding, and CATCs bind and rotate all five periportal triplexes, but notably only about one peri-penton triplex. These results offer insights into the EBV capsid assembly and a mechanism for recruiting cell-regulating factors into the tegument compartment as 'cargoes', and should inform future anti-EBV strategies.
Topics: Amino Acid Sequence; Capsid; Capsid Proteins; Cryoelectron Microscopy; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Imaging, Three-Dimensional; Models, Molecular; Protein Subunits; Structure-Activity Relationship; Virion; Virus Assembly
PubMed: 32719506
DOI: 10.1038/s41564-020-0758-1