Three-dimensional structure determination of capsid of<Emphasis Type="Italic">Aedes albopictus</Emphasis> C6/36 cell densovirus

The three-dimensional structure of capsid ofAedes albopictus C6/36 densovirus was determined to 14-Å resolution by electron cryomicroscopy and computer reconstruction. The triangulation number of the capsid is 1. There are 12 holes in each triangular face and a spike on each 5-fold vertex. The validity of the capsid and nucleic acid densities in the reconstructions was discussed.     

Averaging tens to hundreds of icosahedral particle images to resolve protein secondary structure elements using a Multi-Path Simulated Annealing optimization algorithm

Accurately determining a cryoEM particle's alignment parameters is crucial to high resolution single particle 3-D reconstruction. We developed Multi-Path Simulated Annealing, a Monte-Carlo type of optimization algorithm, for globally aligning the center and orientation of a particle simultaneously. A consistency criterion was developed to ensure the alignment parameters are correct and to remove some bad particles from a large pool of images of icosahedral particles. Without using any a priori model, this procedure is able to reconstruct a structure from a random initial model. Combining the procedure above with a new empirical double threshold particle selection method, we are able to pick tens of best quality particles to reconstruct a subnanometer resolution map from scratch. Using the best 62 particles of rice dwarf virus, the reconstruction reached 9.6A resolution at which four helices of the P3A subunit of RDV are resolved. Furthermore, with the 284 best particles, the reconstruction is improved to 7.9A resolution, and 21 of 22 helices and six of seven beta sheets are resolved.     

Three-dimensional structure determination of capsid of Aedes albopictus C6/36 cell densovirus

Apreviously-unknownviruswasfoundchroni-callyinfectingC6/36cellsthatwereusedtoculturedenguevirus.Afterisolationandpurification,RT,PCRandsequencingwereperformedwithrandomprimers.TheresultsshowthattheviralgenomeisssDNAandis4096ntinlength(GenBankAccessionNo.AY095351).Thenucleicacidsequenceofthispreviously-undescribedvirussharesabout90%iden-titywiththatofAedesaegyptidensonucleosisvirusandtheaminoacidsequencesofthreeproteinsen-codedbythenucleicacidshareabout89%—93%identitieswiththoseofAedesa…     

Dual action of ATP hydrolysis couples lid closure to substrate release into the group II chaperonin chamber

Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.     

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV), a member of the membrane‐containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo‐microscopy (cryo‐EM), we determined the structure of an attenuated vaccine strain, TC‐83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.     

The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle.

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 microm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.     

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.     

A Strategy for Electron Tomographic Data Collection and Crystallographic Reconstruction of Biological Bundles

Structures of highly ordered biological bundles have unique features which call for special experimental and computational methods in electron cryomicroscopy. They can be considered as three-dimensional quasi-crystals and reconstructed using a crystallographic approach. However, they are neither “infinitely” large with respect to the borders of the bundle, nor are they a single unit cell in thickness along the viewing direction. Also, because of their shape, bundles do not generally have a preferred azimuthal orientation, which poses challenges for orientation estimation and refinement. We developed a strategy for recording and processing electron cryomicroscopic images that differs from classical two-dimensional crystalline reconstruction techniques. These developments allowed us to merge data from tomographic tilt series of ice-embedded acrosomal bundles. The goal is to determine accurately amplitudes and phases at the diffraction maxima in terms ofhklindices, and compute a three-dimensional map from the diffraction data.     

Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory disease with a complex genetic component. An association between RA and the human leukocyte antigen (HLA) complex has long been observed in many different populations, and most studies have focused on a direct role for the HLA-DRB1 "shared epitope" in disease susceptibility. We have performed an extensive haplotype analysis, using 54 markers distributed across the entire HLA complex, in a set of 469 multicase families with RA. The results show that, in addition to associations with the DRB1 alleles, at least two additional genetic effects are present within the major histocompatibility complex. One of these lies within a 497-kb region in the central portion of the HLA complex, an interval that excludes DRB1. This genetic risk factor is present on a segment of a highly conserved ancestral A1-B8-DRB1*03 (8.1) haplotype. Additional risk genes may also be present in the HLA class I region in a subset of DRB1*0404 haplotypes. These data emphasize the importance of defining haplotypes when trying to understand the HLA associations with disease, and they clearly demonstrate that such associations with RA are complex and cannot be completely explained by the DRB1 locus.