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The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly [Alpha]-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

A study was conducted to examine the folding of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) in Escherichia coli into the low pH-induced conformation of viral HA2. Crosslinking studies showed that HA2 polypeptide residues 38-175 fold in vivo into a soluble trimer. Crystallography showed that the low pH-induced fold of HA2 is the lowest-energy-state fold of the HA2 polypeptide upon the removal of the receptor-binding chain.

Abstract Background Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs. Results Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5–10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach. Conclusion The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.

Influenza virus hemagglutinin(HA), the most extensively characterized membrane fusion protein, is a homotrimeric type I transmembrane protein. Evidence are provided which state that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion. [ABSTRACT FROM AUTHOR]

Blood-stage malaria vaccine candidates include surface proteins of the merozoite. Antibodies to these proteins may either block essential steps during invasion or render the merozoite susceptible to phagocytosis or complement-mediated degradation. Structural information on merozoite surface proteins complexed to antibodies provides crucial information for knowledge-based vaccine design. The major merozoite surface protein MSP1 is an abundant surface molecule in Plasmodium falciparum. Only a subset of antibodies against MSP119 inhibits invasion (inhibitory antibodies), whereas other antibodies binding to MSP119 have no effect on invasion (neutral antibodies). Here we report on the complex of MSP119 with both inhibitory monoclonal antibody 12.10 and neutral monoclonal antibody 2F10. The complexes were established using both whole IgG’s and Fab fragments, and analysed by dynamic light scattering, electron microscopy and analytical ultra centrifugation. Specific ring structures were formed in the ternary complex with the two antibodies, providing direct evidence of non-overlapping epitopes on MSP119. Mutational studies also indicated that the epitopes of the inhibitory and neutral antibodies are spatially remote. [Copyright &y& Elsevier]