Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization

A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment. DOI: http://dx.doi.org/10.7554/eLife.00435.00

Protein interactions involved in alphavirus assembly

Viruses have to protect their genomes during transport from the infected to an uninfected host cell. This is achieved by forming a protein shell around the nucleic acid. Virus assembly is a complex process that involves interactions with the viral genome, the viral structural proteins and for enveloped viruses a lipid membrane. The assembly mechanisms differ between virus families and are in most cases poorly understood. This thesis describes protein interactions involved in alphavirus assembly. Alphaviruses are among the best-characterised enveloped viruses. They have positive single-stranded RNA genomes encapsidated by 240 copies of the capsid (C) protein, forming a nucleocapsid (NC). This is surrounded by a lipid bilayer derived from the host cell plasma membrane during budding. 80 spike complexes composed of three heterodimers of the envelope proteins, El and E2, traverse the membrane and interact with the nucleocapsid. The capsid protein has an autoprotease activity and cleaves itself from the growing polypeptide chain. Its enzymatic activity is special in that it only works in one reaction. The last Trp residue remains bound to the substrate pocket after cleavage and functions as a built in inhibitor. Analysis of a set of mutants showed that the Trp residue was not only important as an inhibitor but also for correct folding and stability of the capsid protein. The assembly of alphaviral particles depends on the interaction between transmembrane spike proteins and the nucleocapsid. A structural model of the interaction was made and tested by a set of mutants. The interaction was found to involve a conserved Tyr-X-Leu motif in the E2 cytoplasmic tail. The motif bound into a hydrophobic cavity on the capsid protein. The E2 Tyr residue interacted with a Tyr and a Trp residue in the first half of the pocket while the Leu interaction in the other half was less specific according to the model. Modelling of the cavity from different alphaviruses suggest that the cavity has evolved to fit an internal capsid motif rather than the spike. The SFV motif Met-X-Ile was indeed found to be important for nucleocapsid assembly. Mutants of this motif did not form intracellular nucleocapsids. However, virus was released in wild type amounts indicating that NCs could form at the site of budding. A possible switch mechanism for nucleocapsid assembly involving the spike-binding cavity are discussed. The results in this thesis have furthered our knowledge about the mechanisms involved in alphavirus assembly. It is clear that the interactions between the spike and capsid proteins are specific and possess a central role in the assembly process

M-X-I Motif of Semliki Forest Virus Capsid Protein Affects Nucleocapsid Assembly

Alphavirus budding is driven by interactions between spike and nucleocapsid proteins at the plasma membrane. The binding motif, Y-X-L, on the spike protein E2 and the corresponding hydrophobic cavity on the capsid protein were described earlier. The spike-binding cavity has also been suggested to bind an internal hydrophobic motif, M113-X-I115, of the capsid protein. In this study we found that replacement of amino acids M113 and I115 with alanines, as single or double mutations, abolished formation of intracellular nucleocapsids. The mutants could still bud efficiently, but the NCs in the released virions were not stable after removal of the membrane and spike protein layer. In addition to wild-type spherical particles, elongated multicored particles were found at the plasma membrane and released from the host cell. We conclude that the internal capsid motif has a biological function in the viral life cycle, especially in assembly of nucleocapsids. We also provide further evidence that alphaviruses may assemble and bud from the plasma membrane in the absence of preformed nucleocapsids