Interpretation of electron density with stereographic roadmap projections

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between ␤H and ␤I of the core ␤-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity. R ecombinant adeno-associated viruses (rAAVs) are promising viral vectors for gene delivery applications (1, 2). These viruses belong to the single-stranded DNA (ssDNA)-packaging Parvoviridae and genus Dependovirus. Hundreds of AAV genotypes have been sequenced from several mammalian species, and to date 12 serotypes (AAV1 to AAV12) have been defined for the human and nonhuman primate isolates (3-15). The 12 serotypes are classified into eight genetic groups, clades A to F and clonal isolates AAV4 and AAV5, based on antigenic reactivity and sequence similarity (15). The groups are represented by AAV1 to AAV9, with AAV1 and AAV6 belonging to the same clade A because of their high sequence similarity and antigenic cross-reactivity. The representative members share ϳ55 to 99% sequence identity, with AAV4 and AAV5 being the most divergent from each other and from the other members.The linear ssDNA AAV genome, ϳ4.7 kb in length, contains two genes: cap, which encodes the capsid viral proteins (VPs; VP1, ϳ87 kDa; VP2, ϳ73 kDa; VP3, ϳ62 kDa) and rep, which encodes the replication proteins necessary for genome replication and genome packaging. Inverted terminal repeats (ITRs) at the end of the AAV genome, 145 bp in length, are the only essential active sequence required to function as (i) the origin for DNA replication, (ii) the packaging signal, and (iii) integration sites (16)(17)(18)(19). Recombina...

Section: Methodsmentioning

confidence: 99%

Structural Insights into Adeno-Associated Virus Serotype 5

Govindasamy

DiMattia

Gurda

et al. 2013

“…The refined fit of the Fab (correlation coefficient value of 0.743) was used to identify all atoms within 4 Å of the fitted virus structures to generate the Fab-binding footprint. Stereographic projections were done using the program RIVEM (30), and Coulombic representations of the surface were generated using Chimera (28).…”

Section: Methodsmentioning

confidence: 99%

A Strain-Specific Epitope of Enterovirus 71 Identified by Cryo-Electron Microscopy of the Complex with Fab from Neutralizing Antibody

Lee

Cifuente

Ashley

et al. 2013

e Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody. E nterovirus 71 (EV71) infection causes outbreaks of hand, foot, and mouth disease (HFMD), predominantly in the Asia-Pacific region (1, 2). Whereas most EV71 infections are self-limiting, neurological and systemic complications can develop that range from aseptic meningitis to encephalitis and acute flaccid paralysis. Infection can lead to lethal pulmonary edema and heart failure (2), with mortality being especially high in young children under the age of 6 (2, 3). As seasonal outbreaks of HFMD are recurring around the world, development of a vaccine and antiviral therapies for EV71 has become an urgent concern.A member of the Picornaviridae family, EV71 has a nonenveloped, icosahedral capsid comprised of 60 copies of each of four viral structural proteins (VP1 to VP4) (4). Recent studies have solved the structures for three strains of EV71 (MY104 [5], Fuyang [6], and 1095 [7]), demonstrating that EV71 has the general features of picornavirus capsids, including the 5-fold "mesa" and the depression around the mesa called the "canyon" (5-8). Conserved residues VP1-242K and VP1-244K form positively charged patches on the 5-fold mesa (6), and this symmetry-related clustering of positive charges has been suggested as a common mechanism for heparan sulfate binding in enteroviruses (9).Several cellular receptors for EV71 have been identified: scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and heparan sulfate (HS) (10-12). SCARB2, which is expressed on a broad variety of cell types, likely binds to the virus canyon and induces the transition of the virion that is required for uncoating (13-15). PSGL-1, which is expressed exclusively on lymphocytes, binds only specific EV71 strains and supports viral replication in lymphocytes in a PSGL-1-dependent manner (11). According to recent studies, PSGL-1 and HS bind the positively charged patches on the 5-fold mesa of EV71 and provide initial attachment on the cell (12,16,17). We recently found that the PSGL-1 binding phenotype of EV71 strains is regulated b...

“…Residue 129, located in the VP1 unique region, is part of a PLA2 domain in the parvoviruses that is predicted to be located inside the assembled capsid but that later becomes externalized through the 5-fold channel during capsid trafficking through the endocytic pathway. This is (72) of surface residues in a portion of the icosahedral asymmetric unit for crystal structures of AAV1 (A), AAV2 (B), AAV5 (C), and AAV6 (D) are shown. The area occupied by each amino acid residue correlates to surface exposure when the capsid is viewed down an icosahedral 2-fold axis.…”

Section: Vol 84 2010 Structure Of Aav6 12949mentioning

confidence: 99%

Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

Govindasamy

Gurda

et al. 2010

122

137

The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7-and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (␤B to ␤I) ␤-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.