Noroviruses are one of the major causes of nonbacterial gastroenteritis epidemics in humans. Recent studies on norovirus receptors show that different noroviruses recognize different human histo-blood group antigens (HBGAs), and eight receptor binding patterns of noroviruses have been identified. The P domain of the norovirus capsids is directly involved in this recognition. To determine the precise locations and receptor binding modes of HBGA carbohydrates on the viral capsids, a recombinant P protein of a GII-4 strain norovirus, VA387, was cocrystallized with synthetic type A or B trisaccharides. Based on complex crystal structures observed at a 2.0-Å resolution, we demonstrated that the receptor binding site lies at the outermost end of the P domain and forms an extensive hydrogen-bonding network with the saccharide ligand. The A and B trisaccharides display similar binding modes, and the common fucose ring plays a key role in this interaction. The extensive interface between the two protomers in a P dimer also plays a crucial role in the formation of the receptor binding interface.

Noroviruses interact with histo-blood group antigen (HBGA) receptors in a strain-specific manner probably detecting subtle structural differences in the carbohydrate receptors. The specific recognition of types A and B antigens by various norovirus strains is a typical example. The only difference between the types A and B antigens is the acetamide linked to the terminal galactose of the A but not to the B antigen. The crystal structure of the P dimer of a GII-4 norovirus (VA387) bound to types A and B trisaccharides has elucidated the A/B binding site on the capsid but did not explain the binding specificity of the two antigens. In this study, using site-directed mutagenesis, we have identified three residues on the VA387 capsid that are sterically close to the acetamide and are required for binding to A but not B antigens, indicating that the acetamide determines the binding specificity between the A and B antigens. Further mutational analysis showed that a nearby open cavity may also be involved in binding specificity to HBGAs. In addition, a systematic mutational analysis of residues in and around the binding interface has identified a group of amino acids that are required for binding but do not have direct contact with the carbohydrate antigens, implying that these residues may be involved in the structural integrity of the receptor binding interface. Taken together, our study provides new insights into the carbohydrate/capsid interactions which are a valuable complement to the atomic structures in understanding the virus/host interaction and in the future design of antiviral agents.

The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of viruslike particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thinsection transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta-and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta-and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumaviruslike particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. IMPORTANCEComparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway. The Gag polyprotein is the primary retroviral structural protein responsible for orchestrating retrovirus assembly. HIV-1 Gag has arguably been the most extensively studied Gag polyprotein to date (1-7); however, distinct differences in the biology of retroviral Gag proteins have emphasized the importance of comparative analyses in order to gain further insights (8).The Gag polyprotein is functionally conserved across the two retroviral subfamilies (i.e., Orthoretrovirinae and Spumaretrovirinae). While the six genera in the Orthoretrovirinae subfamily (Alpha-, Beta-, Delta-, Epsilon-, Gamma-, and Lentivirus) an...

Double ionization of the helium atom by slow electron impact (E(0)=106 eV) is studied in a kinematically complete experiment. Because of a low excess energy E(exc)=27 eV above the double ionization threshold, a strongly correlated three-electron continuum is realized. This is demonstrated by measuring and calculating the fully differential cross sections for equal energy sharing of the final-state electrons. While the electron emission is dominated by a strong Coulomb repulsion, also signatures of more complex dynamics of the full four-body system are identified.