The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

Abstract

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.

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

“…Now with a very high resolution structure as a yardstick, it is enlightening to analyze the accuracy of prior AAV-DJ structures at 2.8 Å and 4.5 Å, [ 20 , 22 ], the former representing a resolution that is often a goal in the emerging post “resolution revolution” era of cryo-EM, while the latter represents the 5 Å average of structures deposited to EMDB in 2019–2020. It is sobering that the root mean square deviation (RMSD) between the 2.8 Å and 1.56 Å structures is as high as 0.9 Å, given estimates that stereochemical restraints in protein crystallography support accuracy 5–8-fold beyond the nominal experimental resolution [ 31 , 58 , 59 , 60 ].…”

“…Furthermore, the results were achieved with a microscope configuration that has become standard over the last couple of years. Approximately the same number of particles (47,000; 2.6 × 10 6 asymmetric units) were used as in an earlier (2017) 2.8 Å structure determination [ 22 ], in other words, this is a standard data collection regime without extraordinary efforts to maximize the resolution. The results reported here have not depended on the very latest microscope developments that have supported apoferritin structures beyond 1.5 Å [ 48 , 49 ].…”

“…Envelope corrections and resolution, together with the effective EM magnification (at the start), were least-squares refined periodically against the emerging atomic model, using RSRef [ 31 ]. Calibration of the magnification was against the prior 2.8 Å cryo-EM structure of a glycan complex [ 22 ], and ultimately back to an external crystallographic standard, the AAV2 crystal structure [ 24 ]. Resolution is refined by optimizing the parameters of a Butterworth low-pass filter applied to the atomic model, until its density optimally agrees with the experimental reconstruction.…”

“…AAV-DJ is a recombinant chimeric mix of natural serotypes 2, 8 and 9, selected from a randomized library for liver tropism and immune evasion [ 19 ]. Like the natural type-species, AAV-2, it attaches to extracellular heparanoids and became a model system in our laboratory for studying the glycan interactions [ 20 , 21 , 22 ]. Before the cryo-EM “resolution revolution” [ 23 ], studies of viral interactions with di- to penta-saccharides pushed the then-limits, and it was helpful to have the increased EM contrast and superior alignment of recombinantly expressed virus like particles (VLPs) over otherwise identical DNA-containing wild-type viruses.…”

Fondaparinux: Should It Be Studied in Patients with COVID-19 Disease?

“…19 The same result was obtained employing FPX whose structure differs from that of both heparin and heparin sulfate for a terminal o-methyl group but it is quite similar to the sequences prevailing in heparin and heparan sulfate to which several AAVs bind during entry. 20 In other words, FPX is a sulfated molecule whose antiviral properties should be studied in a more extensive way in the future.…”