Structures of enveloped virions determined by cryogenic electron microscopy and tomography : Advances in Virus Research

Enveloped viruses enclose their genomes inside a lipid bilayer which is decorated by membrane proteins that mediate virus entry. These viruses display a wide range of sizes, morphologies and symmetries. Spherical viruses are often isometric and their envelope proteins follow icosahedral symmetry. Filamentous and pleomorphic viruses lack such global symmetry but their surface proteins may display locally ordered assemblies. Determining the structures of enveloped viruses, including the envelope proteins and their protein-protein interactions on the viral surface, is of paramount importance. These structures can reveal how the virions are assembled and released by budding from the infected host cell, how the progeny virions infect new cells by membrane fusion, and how antibodies bind surface epitopes to block infection. In this chapter, we discuss the uses of cryogenic electron microscopy (cryo-EM) in elucidating structures of enveloped virions. Starting from a detailed outline of data collection and processing strategies, we highlight how cryo-EM has been successfully utilized to provide unique insights into enveloped virus entry, assembly, and neutralization.Peer reviewe

A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface

Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.Subject Categories Microbiology, Virology & Host Pathogen Interaction; Immunolog

Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers

There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo–electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 “locks” two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics

Transposons and pathogenicity in Xanthomonas: acquisition of murein lytic transglycosylases by TnXax1 enhances Xanthomonas citri subsp. citri 306 virulence and fitness

Xanthomonas citri subsp. citri 306 (XccA) is the causal agent of type A citrus canker (CC), one of the most significant citriculture diseases. Murein lytic transglycosylases (LT), potentially involved in XccA pathogenicity, are enzymes responsible for peptidoglycan structure assembly, remodeling and degradation. They directly impact cell wall expansion during bacterial growth, septum division allowing cell separation, cell wall remodeling allowing flagellar assembly, bacterial conjugation, muropeptide recycling, and secretion system assembly, in particular the Type 3 Secretion System involved in bacterial virulence, which play a fundamental role in XccA pathogenicity. Information about the XccA LT arsenal is patchy: little is known about family diversity, their exact role or their connection to virulence in this bacterium. Among the LTs with possible involvement in virulence, two paralogue open reading frames (ORFs) (one on the chromosome and one in plasmid pXAC64) are passenger genes of the Tn3 family transposon TnXax1, known to play a significant role in the evolution and emergence of pathogenicity in Xanthomonadales and to carry a variety of virulence determinants. This study addresses LT diversity in the XccA genome and examines the role of plasmid and chromosomal TnXax1 LT passenger genes using site-directed deletion mutagenesis and functional characterization. We identified 13 XccA LTs: 12 belong to families 1A, 1B, 1C, 1D (two copies), 1F, 1G, 3A, 3B (two copies), 5A, 6A and one which is non-categorized. The non-categorized LT is exclusive to the Xanthomonas genus and related to the 3B family but contains an additional domain linked to carbohydrate metabolism. The categorized LTs are probably involved in cell wall remodeling to allow insertion of type 3, 4 and 6 secretion systems, flagellum assembly, division and recycling of cell wall and degradation and control of peptidoglycan production. The TnXax1 passenger LT genes (3B family) are not essential to XccA or for CC development but are implicated in peptidoglycan metabolism, directly impacting bacterial fitness and CC symptom enhancement in susceptible hosts (e.g., Citrus sinensis). This underlines the role of TnXax1 as a virulence and pathogenicity-propagating agent in XccA and suggests that LT acquisition by horizontal gene transfer mediated by TnXax1 may improve bacterial fitness, conferring adaptive advantages to the plant-pathogen interaction process

A Genome Wide Association Study of arabinoxylan content in 2-row spring barley grain

In barley endosperm arabinoxylan (AX) is the second most abundant cell wall polysaccharide and in wheat it is the most abundant polysaccharide in the starchy endosperm walls of the grain. AX is one of the main contributors to grain dietary fibre content providing several health benefits including cholesterol and glucose lowering effects, and antioxidant activities. Due to its complex structural features, AX might also affect the downstream applications of barley grain in malting and brewing. Using a high pressure liquid chromatography (HPLC) method we quantified AX amounts in mature grain in 128 spring 2-row barley accessions. Amounts ranged from ~ 5.2 μg/g to ~ 9 μg/g. We used this data for a Genome Wide Association Study (GWAS) that revealed three significant quantitative trait loci (QTL) associated with grain AX levels which passed a false discovery threshold (FDR) and are located on two of the seven barley chromosomes. Regions underlying the QTLs were scanned for genes likely to be involved in AX biosynthesis or turnover, and strong candidates, including glycosyltransferases from the GT43 and GT61 families and glycoside hydrolases from the GH10 family, were identified. Phylogenetic trees of selected gene families were built based on protein translations and were used to examine the relationship of the barley candidate genes to those in other species. Our data reaffirms the roles of existing genes thought to contribute to AX content, and identifies novel QTL (and candidate genes associated with them) potentially influencing the AX content of barley grain. One potential outcome of this work is the deployment of highly associated single nucleotide polymorphisms markers in breeding programs to guide the modification of AX abundance in barley grain

A highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins

Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7–DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody. There is no licensed vaccine or therapeutic for dengue virus (DENV) infection. Here, the authors show that a highly potent human monoclonal antibody binds to DENV particles in an unusual and very effective way by interacting with three viral envelope proteins