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.

Zika virus (ZIKV) is an arthropod-borne enveloped virus belonging to the Flavivirus genus in the family Flaviviridae, which also includes the human pathogenic yellow fever, dengue, West Nile and tick-borne encephalitis viruses 1 . Flaviviruses have two structural glycoproteins, prM and E (for precursor membrane and envelope proteins, respectively), which form a heterodimer in the endoplasmic reticulum (ER) of the infected cell and drive the budding of spiky immature virions into the ER lumen. These particles transit through the cellular secretory pathway, during which the trans-Golgi-resident protease furin cleaves prM. This processing is required for infectivity, and results in the loss of a large fragment of prM and reorganization of E on the virion surface. The mature particles have a smooth aspect, with 90 E dimers organized with icosahedral symmetry in a 'herringbone' pattern 2,3 .Three-dimensional cryo-electron microscopy (cryo-EM) structures of the mature ZIKV particles have recently been reported to near atomic resolution (3.8 Å) 4,5 , showing that the virus has essentially the same organization as the other flaviviruses of known structure, such as dengue virus (DENV) 3 and West Nile virus 6,7 . The E protein is about 500 amino acids long, with the 400 N-terminal residues forming the ectodomain essentially folded as β-sheets with three domains, named I, II and III, aligned in a row with domain I at the centre. The conserved fusion loop is at the distal end of the rod in domain II, buried at the E dimer interface. At the C terminus, the E ectodomain is followed by the 'stem' , featuring two α-helices lying flat on the viral membrane (the stem helices), which link to two C-terminal transmembrane α-helices. The main distinguishing feature of the ZIKV virion is an insertion within a glycosylated loop of E (the '150' loop), which protrudes from the mature virion surface 4,5 .Flaviviruses have been grouped into serocomplexes based on cross-neutralization studies with polyclonal immune sera 8 . The E protein is the main target of neutralizing antibodies, and is also the viral fusogen; cleavage of prM allows E to respond to the endosomal pH by undergoing a large-scale conformational change that catalyses membrane fusion and releases the viral genome into the cyotosol. Loss of the precursor fragment of prM lets the E protein fluctuate from its tight packing at the surface of the virion, transiently exposing otherwise buried surfaces. One surface exposed by this 'breathing' is the fusionloop epitope (FLE), which is a dominant cross-reactive antigenic site 9 . Although antibodies to this site can protect by complement-mediated mechanisms, as shown in a mouse model for West Nile virus 10 , they are poorly neutralizing and lead to antibody-dependent enhancement 11-15 , thereby aggravating Flavivirus pathogenesis and complicating the development of safe and effective vaccines.We recently reported the functional and structural characterization of a panel of antibodies isolated from patients with dengue disease 13,16 . ...

2Dengue disease is caused by four different flavivirus 1 serotypes, which infect 390 million people yearly with 25% symptomatic cases 2 and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (DENV--2) 3,4 . Structural studies so far have characterized only epitopes recognized by serotype specific human antibodies 5,6 . We recently isolated human antibodies potently neutralizing all four DENV serotypes 7 . Here we describe the X--ray structures of four of these broadly neutralizing antibodies (bnAbs) in complex with the envelope glycoprotein E from DENV--2, revealing that the recognition determinants are at a serotype conserved site at the E dimer interface, including the exposed main chain of the E fusion loop 8 and the two conserved glycan chains.This "E--dimer dependent epitope" (EDE) is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell 9 , explaining its conservation across serotypes and highlighting an Achilles heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.Exposed at the surface of infectious mature DENV particles, protein E is the sole target of neutralizing antibodies. It displays an icosahedral arrangement in which 90 E dimers completely coat the viral surface 10,11 and which is sensitive to the environmental pH. Upon entry of DENV into cells via receptor--mediated endocytosis, the acidic 3 endosomal environment triggers an irreversible fusogenic conformational change in E that leads to fusion of viral and endosomal membranes 1 . The structure of the isolated E dimer has been determined by X--ray crystallography using the soluble ectodomain (sE) 8,12 . Protein E is relatively conserved, displaying about 65% amino acid sequence identity when comparing the most distant DENV serotypes. In particular, there are two conserved N--linked glycosylation sites at positions N67 and N153. To examine its interaction with the antibodies, we selected four highly potent bnAbs identified in the accompanying work: 747(4) A11 and 747 B7 (EDE2 group, requiring glycosylation at position N153 for efficient binding) and 752--2 C8 and 753(3) C10 (EDE1 group, binding regardless of the glycosylation at N153) 7 -referred to as A11, B7, C8 and C10 from hereon. The EDE2 bnAbs were isolated from the same patient (who had a secondary infection with DENV--2), and are somatic variants of the same IgG clone, derived from the IGHV3--74 and IGLV2--23 germ lines. The heavy chain has a very long (26 amino acids, IMGT convention) complementarity--determining region 3 (CDR H3). The EDE1 bnAbs were isolated from different patients and derive from (EDE1 C8, the patient appeared to have a primary infection of undetermined serotype) and IGHV1--3* and IGLV2--14 (EDE1 C10, from a patient with secondary DENV--1 infecti...

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic ‘breathing' of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We here engineer E dimers locked by inter-subunit disulfide bonds, and show by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccine.

Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant (DeltaUL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the DeltaUL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or DeltaUL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

PrPSc forms micrometer long amyloidic strings that can congregate in clusters and webs at the surface of living cells.

Highlights d The C10 Fab's orientation on E dimers allows bivalent IgG binding to each virion raft d C10 binding to DENV2 induces E dimer rearrangement by hitting a spring-loaded segment d Only E dimers with asymmetric environment on DENV2 virions can bind C10 d Bivalent binding to two different E dimers expands C10 neutralization breadth

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication–transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.