Whereas the protein composition and overall shape of several giant virus capsids have been described, the mechanism by which these large capsids assemble remains enigmatic. Here, we present a reconstruction of the capsid of Cafeteria roenbergensis virus (CroV), one of the largest viruses analyzed by cryo-electron microscopy (cryo-EM) to date. The CroV capsid has a diameter of 3,000 Å and a Triangulation number of 499. Unlike related mimiviruses, the CroV capsid is not decorated with glycosylated surface fibers, but features 30 Å-long surface protrusions that are formed by loops of the major capsid protein. Based on the orientation of capsomers in the cryo-EM reconstruction, we propose that the capsids of CroV and related giant viruses are assembled by a newly conceived assembly pathway that initiates at a five-fold vertex and continuously proceeds outwards in a spiraling fashion.Viruses with long dsDNA genomes (>200 kilobases) and large particles (>0.2 µm) are a common occurrence in nature and several new families of giant viruses have been reported in recent years [1][2][3][4][5][6] , which inspired various discussions regarding their evolutionary origin 7-11 . However, whereas rapidly advancing DNA sequencing methods facilitate genomic analysis of giant viruses, structural studies of large viral capsids are lagging behind. In contrast to small and medium-sized capsids, the >200 nm isometric capsids of giant DNA viruses still pose a significant technical challenge for high-resolution methods such as X-ray crystallography and cryo-EM 12 . Other techniques, including atomic force microscopy, scanning electron microscopy, and X-ray free electron lasers, have been used to study giant virus structures, but are unable to provide near-atomic resolution [13][14][15][16] . During the last decade, cryo-EM has become an increasingly powerful tool to determine the structure of virus particles, circumventing the need for crystallization [17][18][19][20] . The resolution of cryo-EM reconstruction of viruses has gradually improved from sub-nanometer to near atomic levels [21][22][23][24][25][26][27][28][29][30] . In addition, combined with X-ray crystallography of purified capsid proteins, it is possible to fit the atomic structures of individual components into the cryo-EM reconstruction map and determine a pseudo-atomic structure [31][32][33][34] . Examples for the successful combination of X-ray crystallography and cryo-EM to determine large DNA virus structures are Paramecium bursaria Chlorella virus 1 (PBCV-1) 35,36 and Chilo iridescent virus (CIV) 37 .Here, we push the limits of cryo-EM application to large virus particles by reconstructing the capsid of Cafeteria roenbergensis virus (CroV) 38 . The giant virus CroV infects the widespread marine zooplankter Cafeteria roenbergensis, a single-celled eukaryote and ecologically important bacterivore 39 . Phylogenetically, CroV is a distant relative of the giant Acanthamoeba polyphaga mimivirus (APMV) 1 and the sole member of the genus Cafeteriavirus in the family Mimivirida...

Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.

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Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication.