Adeno-associated virus (AAV) is a small ssDNA satellite virus that receives wide attention due to its popularity as a safe and effective gene therapy vector. The AAV cell entry receptor (AAVR) for most serotypes is a glycoprotein containing five polycystic kidney disease (PKD) domains with which AAV interacts. AAV serotypes can be classified into three groups: those that interact primarily with PKD1, those whose interactions with PKD2 are stronger, and AAV4-like serotypes whose transduction is AAVR-independent. A phylogenetic analysis of AAVR and paralog KIAA0319 revealed AAVR amino acid variability in the region of PKD1 that is bound by AAV. We hypothesized that the substitution, in all cat-like animals, of a glutamate at a site that is an arginine (R353) in human AAVR may interfere with the binding of clade H AAVs that interact exclusively with PKD1. Analysis of PKD1 mutations, including ELISA, shows that an R353E substitution of glutamate for arginine affects the binding of the clade H AAVs that interact primarily with PKD1.

Adeno-associated viruses (AAV) are among the foremost vectors for in vivo gene therapy. A number of monoclonal antibodies against several serotypes of AAV have previously been prepared. Many are neutralizing, and the predominant mechanisms have been reported as the inhibition of binding to extracellular glycan receptors or interference with some post-entry step. The identification of a protein receptor and recent structural characterization of its interactions with AAV compel reconsideration of this tenet. AAVs can be divided into two families based on which domain of the receptor is strongly bound. Neighboring domains, unseen in the high-resolution electron microscopy structures have now been located by electron tomography, pointing away from the virus. The epitopes of neutralizing antibodies, previously characterized, are now compared to the distinct protein receptor footprints of the two families of AAV. Comparative structural analysis suggests that antibody interference with protein receptor binding might be the more prevalent mechanism than interference with glycan attachment. Limited competitive binding assays give some support to the hypothesis that inhibition of binding to the protein receptor has been an overlooked mechanism of neutralization. More extensive testing is warranted.

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, a high-resolution cryo-electron microscopy structure (2.2 Å) of AAV serotype 4 (AAV4) is presented. The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, AAV12 and AAVrh32.33) 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 1 (PKD1) 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 of AAVR. 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 to be sufficient to exclude binding with PKD1.

ABSTRACTAdeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV’s human cell entry receptor (AAVR) contains Polycystic Kidney Disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA studies, it is shown that AAVGo.1 binds to human AAVR (huAAVR) more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of huAAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure, except for disordering of a neighboring surface loop. Only 4 of the 42 capsid protein sequence differences between AAVGo.1 and AAV5 occur at the PKD1 binding interface. These result in only minor conformational changes in AAVR, including a near rigid domain rotation with maximal displacement of the receptor by ~1 Å. A picture emerges of two classes of AAV with completely different modes of binding to the same AAVR receptor, but within each class atomic interactions are mostly conserved.IMPORTANCEAdeno-Associated Virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor huAAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

Adeno-associated virus (AAV) is the leading vector in emerging treatments of inherited diseases. Higher transduction efficiencies and cellular specificity are required for broader clinical application, motivating investigations of virus-host molecular interactions during cell entry. High-throughput methods are identifying host proteins more comprehensively, with subsequent molecular studies revealing unanticipated complexity and serotype specificity. Cryogenic electron microscopy (cryo-EM) provides a path towards structural details of these sometimes heterogeneous virus-host complexes, and is poised to illuminate more fully the steps in entry. Here presented, is progress in understanding the distinct steps of glycan attachment, and receptor-mediated entry/trafficking. Comparison with structures of antibody complexes provides new insights on immune neutralization with implications for the design of improved gene therapy vectors.

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.

Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery vector in emerging gene therapy treatments for inherited disorders, so its structure and molecular interactions with human hosts are of intense interest. A wide array of electron microscopic approaches have been used to visualize the virus and its complexes, depending on the scientific question, technology available, and amenability of the sample. Approaches range from subvolume tomographic analyses of complexes with large and flexible host proteins to detailed analysis of atomic interactions within the virus and with small ligands at resolutions as high as 1.6 Å. Analyses have led to the reclassification of glycan receptors as attachment factors, to structures with a new-found receptor protein, to identification of the epitopes of antibodies, and a new understanding of possible neutralization mechanisms. AAV is now well-enough characterized that it has also become a model system for EM methods development. Heralding a new era, cryo-EM is now also being deployed as an analytic tool in the process development and production quality control of high value pharmaceutical biologics, namely AAV vectors.

Journal Article Advancing High-resolution Imaging of Human Viruses in Liquid Get access GM Jonaid, GM Jonaid Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, USAMaterials Research Institute, Pennsylvania State University, University Park, PA, USA Search for other works by this author on: Oxford Academic Google Scholar William J Dearnaley, William J Dearnaley Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, USAMaterials Research Institute, Pennsylvania State University, University Park, PA, USA Search for other works by this author on: Oxford Academic Google Scholar Samantha Berry, Samantha Berry Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, USAMaterials Research Institute, Pennsylvania State University, University Park, PA, USA Search for other works by this author on: Oxford Academic Google Scholar Jennifer Gray, Jennifer Gray Materials Research Institute, Pennsylvania State University, University Park, PA, USA Search for other works by this author on: Oxford Academic Google Scholar Michael Spilman, Michael Spilman Applications team, Direct Electron, LP, San Diego, CA, USA Search for other works by this author on: Oxford Academic Google Scholar Madeline J Dukes, Madeline J Dukes Application Scientist, Protochips, Inc., Morrisville, NC, USA Search for other works by this author on: Oxford Academic Google Scholar Deborah F Kelly Deborah F Kelly Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, USAMaterials Research Institute, Pennsylvania State University, University Park, PA, USA Corresponding author: Debkelly@psu.edu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Page 100, https://doi.org/10.1017/S1431927622001301 Published: 01 August 2022

With 150 clinical trials for 30 diseases under way, 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.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.

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.