scholarly journals Cryo-EM Structure of Adeno-associated virus-4 at 2.2 Å resolution

2022 ◽  
Author(s):  
Grant M Zane ◽  
Mark A Silveria ◽  
Nancy L Meyer ◽  
Tommi A White ◽  
Michael S Chapman
Keyword(s):  
Electron Microscopy ◽  
Gene Therapy ◽  
Variable Region ◽  
Cell Receptor ◽  
Polycystic Kidney ◽  
Adeno Associated Virus ◽  
Variable Regions ◽  
Virus Receptor ◽  
Cryo Electron Microscopy ◽  
Viral Surface

Adeno-associated virus (AAV) is the vector of choice for several approved gene therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, we present a high-resolution cryo-electron microscopy structure (2.2 Å) for AAV serotype 4 (AAV4). The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, 12 and rh32.33) is unknown. Other AAVs interact with the same cell receptor, Adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like [PKD]-1 domain of AAVR while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR, provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear sufficient to exclude binding with PKD1.

scholarly journals Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

Structure ◽  
2012 ◽  
Vol 20 (8) ◽  
pp. 1310-1320 ◽  
Author(s):  
Thomas F. Lerch ◽  
Jason K. O'Donnell ◽  
Nancy L. Meyer ◽  
Qing Xie ◽  
Kenneth A. Taylor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gene Therapy ◽  
Gene Therapy Vector ◽  
Cryo Electron Microscopy

scholarly journals Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy

2019 ◽  
Vol 30 (12) ◽  
pp. 1449-1460
Author(s):  
Suriyasri Subramanian ◽  
Anna C. Maurer ◽  
Carol M. Bator ◽  
Alexander M. Makhov ◽  
James F. Conway ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adeno Associated Virus ◽  
Cryo Electron Microscopy ◽  
Virus Capsids

scholarly journals Influence of T cell receptor V alpha expression on Mlsa superantigen-specific T cell responses.

1992 ◽  
Vol 175 (5) ◽  
pp. 1405-1408 ◽  
Author(s):  
M S Vacchio ◽  
O Kanagawa ◽  
K Tomonari ◽  
R J Hodes
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Variable Region ◽  
Cell Receptor ◽  
Variable Regions ◽  
Peripheral T Cells ◽  
Multiple Variable ◽  
Alpha Beta

Recognition of conventional foreign antigen by T cells is determined by the expression of multiple variable regions of both alpha and beta chains of the T cell receptor (TCR) alpha/beta heterodimer. In contrast, there exists a class of antigens that appears to interact with the TCR alpha/beta heterodimer through the variable region on the beta chain (V beta), independent of other TCR components, a property that has led to their designation as superantigens. The goal of the present study was to analyze V alpha use in V beta 6+ T cells responsive to the superantigen, Mlaa. Results indicate that while deletion of T cells expressing V beta 6 in Mlsa-expressing mice is essentially complete and therefore appears to occur regardless of V alpha usage, in vitro Mlsa stimulation of T cells from Mlsa-negative mice results in significant skewing of V alpha use among responding V beta 6+ T cells. This indicates that V alpha expression influences recognition of the superantigen, Mlsa by mature peripheral T cells.

scholarly journals Adeno-Associated Virus (AAV-DJ)—Cryo-EM Structure at 1.56 Å Resolution

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1194
Author(s):  
Qing Xie ◽  
Craig K. Yoshioka ◽  
Michael S. Chapman
Keyword(s):  
Electron Microscopy ◽  
Hydrogen Bond ◽  
Viral Vector ◽  
A Priori ◽  
Viral Assembly ◽  
X Ray Diffraction ◽  
Adeno Associated Virus ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Structural Virology

Adeno-associated virus is the leading viral vector for gene therapy. AAV-DJ is a recombinant variant developed for tropism to the liver. The AAV-DJ structure has been determined to 1.56 Å resolution through cryo-electron microscopy (cryo-EM). Only apoferritin is reported in preprints at 1.6 Å or higher resolution, and AAV-DJ nearly matches the highest resolutions ever attained through X-ray diffraction of virus crystals. However, cryo-EM has the advantage that most of the hydrogens are clear, improving the accuracy of atomic refinement, and removing ambiguity in hydrogen bond identification. Outside of secondary structures where hydrogen bonding was predictable a priori, the networks of hydrogen bonds coming from direct observation of hydrogens and acceptor atoms are quite different from those inferred even at 2.8 Å resolution. The implications for understanding viral assembly mean that cryo-EM will likely become the favored approach for high resolution structural virology.

scholarly journals The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1326
Author(s):  
Mark A. Silveria ◽  
Edward E. Large ◽  
Grant M. Zane ◽  
Tommi A. White ◽  
Michael S. Chapman
Keyword(s):  
Gene Therapy ◽  
Binding Sites ◽  
Polycystic Kidney ◽  
Adeno Associated Virus ◽  
Gene Therapies ◽  
The Us ◽  
Gene Therapy Vectors ◽  
Cellular Entry ◽  
Resolution Structure

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

scholarly journals Highly sensitive and unbiased approach for elucidating antibody repertoires

2016 ◽  
Vol 113 (28) ◽  
pp. 7846-7851 ◽  
Author(s):  
Sherry G. Lin ◽  
Zhaoqing Ba ◽  
Zhou Du ◽  
Yu Zhang ◽  
Jiazhi Hu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germ Line ◽  
Variable Region ◽  
Cell Receptor ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Variable Regions ◽  
Antibody Repertoires ◽  
B Lineage

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

scholarly journals Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

2022 ◽  
Author(s):  
Guiqing Hu ◽  
Mark A Silveria ◽  
Michael S Chapman ◽  
Scott M Stagg
Keyword(s):  
Electron Microscopy ◽  
Gene Therapy ◽  
Viral Entry ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Fold Axis ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Molecular Virology ◽  
Cryo Electron Tomography

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE: With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

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