Atomic Resolution Structures of Human Bufaviruses Determined by Cryo-Electron Microscopy

Abstract

Bufavirus strain 1 (BuV1), a member of the Protoparvovirus genus of the Parvoviridae, was first isolated from fecal samples of children with acute diarrhea in Burkina Faso. Since this initial discovery, BuVs have been isolated in several countries, including Finland, the Netherlands, and Bhutan, in pediatric patients exhibiting similar symptoms. Towards their characterization, the structures of virus-like particles of BuV1, BuV2, and BuV3, the current known genotypes, have been determined by cryo-electron microscopy and image reconstruction to 2.84, 3.79, and 3.25 Å, respectively. The BuVs, 65–73% identical in amino acid sequence, conserve the major viral protein, VP2, structure and general capsid surface features of parvoviruses. These include a core β-barrel (βB-βI), α-helix A, and large surface loops inserted between these elements in VP2. The capsid contains depressions at the icosahedral 2-fold and around the 5-fold axes, and has three separated protrusions surrounding the 3-fold axes. Structure comparison among the BuVs and to available parvovirus structures revealed capsid surface variations and capsid 3-fold protrusions that depart from the single pinwheel arrangement of the animal protoparvoviruses. These structures provide a platform to begin the molecular characterization of these potentially pathogenic viruses.

High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors

“…Individual AAV5-HL2476 particle images were manually extracted from the micrographs using the RobEM software package (35) (http://cryoEM.ucsd .edu/programs.shtm). Preprocessing of the selected images (normalization and apodization) and estimation of the defocus level of each micrograph were performed using the AUTOPP subroutine (options F, O, and 3X) of AUTO3DEM as previously described (35,36). The ab initio random-model computation procedure in AUTO3DEM was used to generate a starting model of the virus-Fab complex at ϳ30-Å resolution from 100 particle images (35).…”

“…This map was used to initiate full orientation and origin determination and refinement of the entire set of images with AUTO3DEM utilizing the gold standard protocol. Corrections to compensate for the effects of phase reversals in the microscope contrast-transfer function for each micrograph were performed as previously described, without amplitude corrections (36). An inverse B-factor (temperature factor) value of 1/25 Å 2 was applied to sharpen the high-resolution features of the resulting final map, followed by visual inspection in the Coot and Chimera programs (37,38).…”

Protease-Activatable Adeno-Associated Virus Vector for Gene Delivery to Damaged Heart Tissue

“…The exceptions were acidic amino acids, as expected, because these residues are known to be sensitive to electron beam-induced radiation damage (data not shown). 23 A superposition of the VP3 3D model generated for L001 onto the AAV9 structure shows the position of the peptide insertion (see broken rectangular box in Figure 1F). The ordered VP3 structure conserved the eight-stranded anti-parallel b-barrel core, with the BIDG sheet facing the virus interior and the aA helix forming the wall of the 2-fold axes, as reported for other parvovirus structures ( Figure 1F).…”

Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach

“…This map was used to initiate full orientation and origin determination and refinement of the entire set of images with AUTO3DEM utilizing the gold standard protocol. Corrections to compensate for the effects of phase reversals in the microscope contrast-transfer function for each micrograph were performed as recently described, without amplitude corrections (28). The resolutions of the final reconstructions were calculated on the basis of a Fourier shell correlation (FSC, 0.5)…”