Combined approaches to study virus structures.

A virus particle must work as a safe box for protecting its genome, but at the same time it has to undergo dramatic conformational changes in order to preserve itself by propagating in a cell infection. Thus, viruses are miniaturized wonders whose structural complexity requires them to be investigated by a combination of different techniques that can tackle both static and dynamic processes. In this chapter we will illustrate how major structural techniques such as X-ray crystallography and electron microscopy have been and can be combined with other techniques to determine the structure of complex viruses. The power of these hybrid method approaches are revealed through the various examples provided.

Structural characterization of the tetrameric form of the major cat allergen Fel d 1.

Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.

Crystal structures of H-2Db in complex with the LCMV-derived peptides GP92 and GP392 explain pleiotropic effects of glycosylation on antigen presentation and immunogenicity.

Post-translational modifications significantly broaden the epitope repertoire for major histocompatibility class I complexes (MHC-I) and may allow viruses to escape immune recognition. Lymphocytic choriomeningitis virus (LCMV) infection of H-2b mice generates CD8+ CTL responses directed towards several MHC-I-restricted epitopes including the peptides GP92 (CSANNSHHYI) and GP392 (WLVTNGSYL), both with a N-glycosylation site. Interestingly, glycosylation has different effects on the immunogenicity and association capacity of these two epitopes to H-2Db. To assess the structural bases underlying these functional results, we determined the crystal structures of H-2Db in complex with GP92 (CSANNSHHYI) and GP392 (WLVTNGSYL) to 2.4 and 2.5 Å resolution, respectively. The structures reveal that while glycosylation of GP392 most probably impairs binding, the glycosylation of the asparagine residue in GP92, which protrudes towards the solvent, possibly allows for immune escape and/or forms a neo-epitope that may select for a different set of CD8 T cells. Altogether, the presented results provide a structural platform underlying the effects of post-translational modifications on epitope binding and/or immunogenicity, resulting in viral immune escape.

Three-dimensional visualization of forming Hepatitis C virus-like particles by electron-tomography.

Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of wt-HCV and explain the size-heterogeneity of its particles.

Design of agonistic altered peptides for the robust induction of CTL directed towards H-2Db in complex with the melanoma-associated epitope gp100.

Immunogenicity of tumor-associated antigens (TAA) is often weak because many TAA are autoantigens for which the T-cell repertoire is sculpted by tolerance mechanisms. Substitutions at main anchor positions to increase the complementarity between the peptide and the MHC class I (MHC-I) binding cleft constitute a common procedure to improve binding capacity and immunogenicity of TAA. However, such alterations are tailored for each MHC-I allele and may recruit different CTL specificities through conformational changes in the targeted peptides. Comparative analysis of substituted melanoma-differentiation antigen gp100 in complex with H-2D(b) revealed that combined introduction of glycine and proline residues at the nonanchor positions 2 and 3, respectively, resulted in an agonistic altered peptide with dramatically enhanced binding affinity, stability, and immunogenicity of this TAA. Peptide vaccination using the p2Gp3P-altered peptide version of gp100 induced high frequencies of melanoma-specific CTL in the endogenous CD8+ repertoire. Crystal structure analysis of MHC/peptide complexes revealed that the conformation of the modified p2Gp3P-peptide was similar to the wild-type peptide, and indicated that this mimotope was stabilized through interactions between peptide residue p3P and the tyrosine residue Y159 that is conserved among most known MHC-I molecules throughout mammalian species. Our results may provide an alternative approach to enhance MHC stabilization capacity and immunogenicity of low-affinity peptides for induction of robust tumor-specific CTL.

Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in parallel with increased gp120 net charge.

To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations between increased Env net charge and lowered CD4(+) T cell counts, enhanced viral fitness, reduced sensitivity to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5 HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness.