This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

The crystallographically determined structure of a soluble fragment from the major envelope protein of a flavivirus reveals an unusual architecture. The flat, elongated dimer extends in a direction that would be parallel to the viral membrane. Residues that influence binding of monoclonal antibodies lie on the outward-facing surface of the protein. The clustering of mutations that affect virulence in various flaviviruses indicates a possible receptor binding site and, together with other mutational and biochemical data, suggests a picture for the fusion-activating, conformational change triggered by low pH.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

BackgroundA chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis.Methods and FindingsWe report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector.ConclusionsThe unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years. CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor into E3 and E2, is responsible for receptor binding, and E1 for membrane fusion. In the context of a concerted multidisciplinary effort to understand the biology of CHIKV, here we report the crystal structures of the precursor p62-E1 heterodimer and of the mature E3-E2-E1 glycoprotein complexes. The resulting atomic models allow the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general. This combination yields a detailed picture of the functional architecture of the 25 MDa alphavirus surface glycoprotein shell. Together with the accompanying report on the structure of the Sindbis virus E2-E1 heterodimer at acidic pH (ref. 3), this work also provides new insight into the acid-triggered conformational change on the virus particle and its inbuilt inhibition mechanism in the immature complex.

Graphical Abstract Highlights d MERS-CoV/SARS-CoV S composite glycan shields analyzed by cryo-EM and mass spectrometry d Structures of MERS-CoV/SARS-CoV S with neutralizing antibodies from survivors d LCA60 inhibits receptor binding by interacting with MERS-CoV S protein/glycans d S230 blocks receptor binding and triggers fusogenic rearrangements via functional mimicry In Brief Structural analysis of the SARS-CoV S and MERS-CoV S glycoproteins in complex with neutralizing antibodies from human survivors sheds light into the mechanisms of membrane fusion and neutralization Walls et al., SUMMARYRecent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue, a flavivirus closely related to ZIKV. Here we investigate the serological crossreaction between the two viruses. Dengue immune plasma substantially crossreacted with ZIKV and could drive antibody-dependent enhancement of ZIKV infection. Using a panel of human anti-dengue monoclonal antibodies we showed that most antibodies reacting to dengue envelope protein also reacted to ZIKV. Antibodies to linear epitopes including the immunodominant fusion loop epitope while able to bind ZIKV could not neutralize the virus but instead promoted ADE. These data indicate that dengue immunity may drive higher ZIKV replication and have clear implications for disease pathogenesis and future ZIKV and dengue vaccine programs.

The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.