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.
High-resolution cryogenic electron microscopy (cryo-EM) structures detail adeno-associated virus (AAV) receptor protein binding interactions.AAV’s glycan 'primary receptors' are recharacterized as 'attachment factors'.AAV5-like viruses interact with a different domain of the receptor using a binding site that is distinct from that of other AAVs.The epitopes of many AAV-neutralizing antibodies overlap with receptor-binding sites on the capsid.Future structural focus will be on AAV receptor-independent pathways and additional steps in entry and trafficking.
Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the ...discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.
Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotype-promiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family.
Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune ...neutralization. Interactions of AAV with its cellular receptor, AAVR, are key to understanding cell-entry and trafficking with the rigor needed to engineer tissue-specific vectors.
-electron tomography shows ordered binding of part of the flexible receptor to the viral surface, with distal domains in multiple conformations. Regions of the virus and receptor in close physical proximity can be identified by cross-linking/mass spectrometry.
-electron microscopy with a two-domain receptor fragment reveals the interactions at 2.4 Å resolution. AAVR binds between AAV's spikes on a plateau that is conserved, except in one clade whose structure is AAVR-incompatible. AAVR's footprint overlaps the epitopes of several neutralizing antibodies, prompting a re-evaluation of neutralization mechanisms. The structure provides a roadmap for experimental probing and manipulation of viral-receptor interactions.
AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to ...in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.
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► The 4.5 Åcryo-EM structure of AAV-DJ fully resolves the polypeptide backbone ► Liver tropism selected for in AAV-DJ has not changed the heparin binding site ► Changed conformation in an antigenic loop blocks binding of a neutralizing mAb ► Changed in vivo tropism may result from changed immune interactions
AAV-DJ is an engineered adeno-associated virus (AAV) vector and a leading candidate for liver gene therapy. Here, Lerch et al. report a 4.5Å resolution cryo-EM structure of AAV-DJ, revealing structural features that underscore its unique phenotype. The reconstruction exemplifies potential of cryo-EM to analyze fine structural changes.
Inhibition of striated muscle contraction at resting Ca2+ depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within ...TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core—and thus tropomyosin—is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca2+-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca2+ was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin—presumably including Ip—are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin.
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•Cardiac troponin I C-terminal mobile domain was assayed by Ca2+-regulated motility.•In one construct Md was partially decoupled from Tn by a short flexible linker.•In another construct Md was eliminated entirely.•Md primarily stabilizes off state of thin filament regulatory units at low Ca2+.•Confirmed Md is not responsible for superactivation of actomyosin cycling by Tn.
Abstract Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests ...that receptor-attachment is mediated by four residues: Arg576 , Lys493 , Lys459 and Lys531 , defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459 . Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2's neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality.
Real-time label-free electrical detection of proteins, including cardiac troponin (cTn), is demonstrated using functionalized SnO₂ nanobelt field-effect transistors (FETs) with integrated ...microfluidics. Selective biomolecular functionalization of the active SnO₂ nanobelt channel and effective passivation of the substrate surface were realized and verified through fluorescence microscopy. The validation/verification of the sensing scheme was initially demonstrated via detection of biotin-streptavidin binding: devices with single biotinylated SnO₂ nanobelts showed pronounced conductance changes in response to streptavidin binding. Importantly, the pH-dependence of the conductance changes was fully consistent with the charged states of streptavidin at different pH. Moreover, the specificity of the sensors' electrical responses was confirmed by co-labeling with quantum dots. Finally, the sensing platform was successfully applied for detection of the cardiac troponin I (cTnI) subunit within cTn, a clinically important protein marker for myocardial infarction.
Independence is the ultimate goal for students with disabilities, including secondary students with autism. One avenue targeted for increasing independence and decreasing prompt-dependency is through ...self-monitoring. In this study, investigators sought to determine whether a difference exists in levels of task independence when three students with autism complete food preparation tasks while self-monitoring using a low-tech treatment (paper/pencil) and high-tech treatment (iPad). Although both interventions decreased the need for prompting thereby increasing independence, students needed less assistance when using the iPad. Students also maintained their levels of independence in food preparation following summer vacation. Social validity interviews indicated students preferred self-monitoring with the iPad over the paper/pencil.
Goltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and ...skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants.
We report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes.
Our combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS.
Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a ...hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ's directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.