Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc.

Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic ß-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.

An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission.

UNLABELLED: Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission. CONCLUSION: This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission.

HMGB1 protein binds to influenza virus nucleoprotein and promotes viral replication.

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.

Functional cloning by phage display.

This review focusses on the isolation of proteins from genomic or cDNA expression products libraries displayed on phage. The use of phage display is highlighted for the characterization of binding proteins with diverse biological functions. Phage display is compared with another strategy, the yeast two-hybrid method. The combination of both strategies is especially powerful to eliminate false positives and to get information on the biochemical functions of proteins.

Degeneracy in the genetic code and its symmetries by base substitutions.

Degeneracy in the genetic code is known to minimise the deleterious effects of the most frequent base substitutions: transitions at the third base of codons are generally synonymous substitutions. Transversions that alter degeneracy were reported by Rumer. Here the other transversions are shown to leave invariant degeneracy when applied to the first base of codons. As a summary, degeneracy is considered with respect to all three types of base substitutions, the transitions and the two types of transversions. The symmetries of degeneracy by base substitutions are independent of the representation of the genetic code and discussed with respect to the quasi-universality of the genetic code.

A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases.

In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3' end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3-Jun/Fos-Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.

In vitro selection for enzymatic activity: a model study using adenylate cyclase.

An in vitro enzyme selection that can, in principle, be generalised to most chemical reactions, is described. It makes use of filamentous phage display and of a tailor-made antibody fragment directed against the reaction product. The conversion of ATP into 3',5'-cyclic AMP catalysed by Bordetella pertussis adenylate cyclase is taken as an example.

How to broaden enzyme substrate specificity: strategies, implications and applications.

For identification of mutations associated with the broadening of enzyme substrate specificity, three strategies, including directed enzyme evolution, are described for selected examples. Implications concerning enzyme models are highlighted. Applications to the field of biocatalysis are discussed. A bidimensional map for the classification of enzyme activities is suggested so as to improve genome annotations.

Efficient display of two enzymes on filamentous phage using an improved signal sequence.

Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50- fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.

Directed enzyme evolution and selections for catalysis based on product formation.

Enzyme engineering by molecular modelling and site-directed mutagenesis can be remarkably efficient. Directed enzyme evolution appears as a more general strategy for the isolation of catalysts as it can be applied to most chemical reactions in aqueous solutions. Selections, as opposed to screening, allow the simultaneous analysis of protein properties for sets of up to about 10(14) different proteins. These approaches for the parallel processing of molecular information 'Is the protein a catalyst?' are reviewed here in the case of selections based on the formation of a specific reaction product. Several questions are addressed about in vivo and in vitro selections for catalysis reported in the literature. Can the selection system be extended to other types of enzymes? Does the selection control regio- and stereo-selectivity? Does the selection allow the isolation of enzymes with an efficient turnover? How should substrates be substituted or mimicked for the design of efficient selections while minimising the number of chemical synthesis steps? Engineering sections provide also some clues to design selections or to circumvent selection biases. A special emphasis is put on the comparison of in vivo and in vitro selections for catalysis.

A method to predict edge strands in beta-sheets from protein sequences.

There is a need for rules allowing three-dimensional structure information to be derived from protein sequences. In this work, consideration of an elementary protein folding step allows protein sub-sequences which optimize folding to be derived for any given protein sequence. Classical mechanics applied to this system and the energy conservation law during the elementary folding step yields an equation whose solutions are taken over the field of rational numbers. This formalism is applied to beta-sheets containing two edge strands and at least two central strands. The number of protein sub-sequences optimized for folding per amino acid in beta-strands is shown in particular to predict edge strands from protein sequences. Topological information on beta-strands and loops connecting them is derived for protein sequences with a prediction accuracy of 75%. The statistical significance of the finding is given. Applications in protein structure prediction are envisioned such as for the quality assessment of protein structure models.

Tailor-made biocatalysts: combining thermodynamics, organic synthesis, molecular biology, biochemistry and microbiology for the design of enzyme selections.

A general strategy for the isolation of catalysts for given chemical reactions was designed. A FIRST LINK BETWEEN GENES AND THEIR CORRESPONDING PROTEINS WAS ESTABLISHED BY PHAGE DISPLAY: using Darwin's principles on evolution based on selection and amplification, rare protein molecules can then be selected for function from a large repertoire prior to their characterization by sequencing of their genes. A second link was created between enzymes and their products. By making use of the chelate effect and of Inovirus particles as a chemical, affinity chromatography for the reaction product is then sufficient to isolate among 10(6) to 10(11) proteins and their genes, the rare ones coding for catalysts of interest. The strategy for the parallel processing of information on the catalytic activity of variants from a large protein repertoire is highlighted in this review.

The genetic code and its optimization for kinetic energy conservation in polypeptide chains.

Why is the genetic code the way it is? Concepts from fields as diverse as molecular evolution, classical chemistry, biochemistry and metabolism have been used to define selection pressures most likely to be involved in the shaping of the genetic code. Here minimization of kinetic energy disturbances during protein evolution by mutation allows an optimization of the genetic code to be highlighted. The quadratic forms corresponding to the kinetic energy term are considered over the field of rational numbers. Arguments are given to support the introduction of notions from basic number theory within this context. The observations found to be consistent with this minimization are statistically significant. The genetic code may well have been optimized according to energetic criteria so as to improve folding and dynamic properties of polypeptide chains.

A rationale for the symmetries by base substitutions of degeneracy in the genetic code.

The first symmetry by base substitutions of degeneracy in the genetic code was described by Rumer (1966) and the other symmetries were identified later by Jestin (2006) and Jestin and Soulé (2007). Here, a rationale accounting for these symmetries is reported. The number of non-synonymous substitutions over the replicated coding sequence is written as a function of the substitution matrix, whose elements are the number of substitutions from any codon to any other codon. The p-adic distance used as a similarity measure and applied to this matrix is shown to be biologically relevant. The rationale indicates that symmetries by base substitutions of degeneracy in the genetic code are symmetries of the measures of the number of non-synonymous substitutions for sets of synonymous codons.

Characterisation of a DNA polymerase highly mutated along the template binding interface.

Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant's broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.

Streptococcus agalactiae DNA polymerase I is an efficient reverse transcriptase.

Genome annotations result generally from large sequence alignments by bioinformatics. Large scale biochemical data are more difficult to obtain. They derive for example from directed protein evolution experiments by selection. A previously reported directed enzyme evolution experiment by in vitro selection of Stoffel fragment variants of Taq DNA polymerase I was used here to predict the activities of Streptococcus agalactiae DNA polymerase I. The reverse transcriptase activity of S. agalactiae DNA polymerase I was measured and the kinetic parameters for this RNA-dependent DNA polymerase are given. RNA-templated DNA repair is suggested as a possible biological function for this biochemical activity.