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Importance: All enveloped viruses enter cells by fusing their envelope with a target cell membrane while avoiding premature fusion with membranes of the producer cell-the latter being particularly important for viruses that bud at internal membranes. Flaviviruses bud in the endoplasmic reticulum, are transported through the TGN to reach the external milieu, and enter other cells via receptor-mediated endocytosis. The trigger for membrane fusion is the acidic environment of early endosomes, which has a similar pH to the TGN of the producer cell. The viral particles therefore become activated to react to mildly acidic pH only after their release into the neutral pH extracellular environment. Our study shows that for yellow fever virus (YFV), the mechanism of activation involves actively knocking out the fusion brake (protein pr) through a localized conformational change of the envelope protein upon exposure to the neutral pH external environment. Our study has important implications for understanding the molecular mechanism of flavivirus fusion activation in general and points to an alternative way of interfering with this process as an antiviral treatment., Competing Interests: The authors declare no conflict of interest.

Numerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp6 12 gp15 12 gp16 6 ) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit β-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage., (© 2022. The Author(s).)

The flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E) 2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E) 2 dimer at acidic pH reveals the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged pr-binding pocket at the E dimer interface, inducing (prM/E) 2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation., (© 2022. The Author(s).)

The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design., Competing Interests: Declaration of interests F.A.R. is a board member and shareholder of EureKARE and MELETIUS Therapeutics. G.R.S. is member of the GSK Vaccines Scientific Advisory Board and a founding shareholder of RQ Biotechnology. G.R.S., F.A.R., P.G.-C., M.-C.V, A.R., S.D., and J.M. are authors in a patent concerning EDE mAbs, including C8 and C10:US20180037611A1 (2018): Anti-dengue vaccines and antibodies. G.R.S., F.A.R., M.-C.V., A.R., and J.M. are authors of patent CA3066488A1 (2017): Neutralising antibody against dengue for use in a method of prevention and/or treatment., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod-like E subunits bend into "hairpins" to bring the fusion loops next to the C-terminal transmembrane (TM) anchors, with the TM-proximal "stem" element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E-protein trimers from the tick-borne encephalitis flavivirus with sequential stem truncations. Our thermostability and antibody-binding data suggest that the stem "zipper" ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C-terminally truncated upstream the CS, which, unlike fully stem-zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important "breathing" intermediates in the E-protein-driven membrane fusion process., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two α-helical turns downstream a stutter in the central coiled-coil helix - precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the "SOSIP" mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form. These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Zika and dengue viruses belong to the Flavivirus genus, a close group of antigenically related viruses that cause significant arthropod-transmitted diseases throughout the globe. Although infection by a given flavivirus is thought to confer lifelong protection, some of the patient's antibodies cross-react with other flaviviruses without cross-neutralizing. The original antigenic sin phenomenon may amplify such antibodies upon subsequent heterologous flavivirus infection, potentially aggravating disease by antibody-dependent enhancement (ADE). The most striking example is provided by the four different dengue viruses, where infection by one serotype appears to predispose to more severe disease upon infection by a second one. A similar effect was postulated for sequential infections with Zika and dengue viruses. In this review, we analyze the molecular determinants of the dual antibody response to flavivirus infection or vaccination in humans. We highlight the role of conserved partially cryptic epitopes giving rise to cross-reacting and poorly neutralizing, ADE-prone antibodies. We end by proposing a strategy for developing an epitope-focused vaccine approach to avoid eliciting undesirable antibodies while focusing the immune system on producing protective antibodies only., (© 2017 Institut Pasteur. Published under the terms of the CC BY NC ND 4.0 license.)

Conserved across the family Herpesviridae , glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gamma-herpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses. IMPORTANCE Herpesviruses are common human and animal pathogens that infect cells by entering via fusion of viral and cellular membranes. Central to the membrane fusion event is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself in the target membrane via two polypeptide segments called fusion loops (FLs). The molecular details of how gB FLs insert into the lipid bilayer have not been described. Here, we provide structural and functional data regarding key FL residues of gB from pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gBs from all herpesviruses with target membranes are established., (Copyright © 2017 American Society for Microbiology.)

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.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Most arthropod-borne viruses (arboviruses), perpetuated by alternation between a vertebrate host and an insect vector, are likely to emerge through minor genetic changes enabling the virus to adapt to new hosts. In the past decade, chikungunya virus (CHIKV; Alphavirus, Togaviridae) has emerged on La Réunion Island following the selection of a unique substitution in the CHIKV E1 envelope glycoprotein (E1-A226V) of an East-Central-South African (ECSA) genotype conferring a higher transmission rate by the mosquito Aedes albopictus. Assumed to have occurred independently on at least four separate occasions, this evolutionary convergence was suspected to be responsible for CHIKV worldwide expansion. However, assumptions on CHIKV emergence were mainly based on viral genetic changes and the role of the mosquito population quasispecies remained unexplored. Here we show that the nature of the vector population is pivotal in selecting the epidemic CHIKV. We demonstrate using microsatellites mosquito genotyping that Ae. albopictus populations are genetically differentiated, contributing to explain their differential ability to select the E1-226V mutation. Aedes albopictus, newly introduced in Congo coinciding with the first CHIKV outbreak, was not able to select the substitution E1-A226V nor to preferentially transmit a CHIKV clone harboring the E1-226V as did Ae. albopictus from La Réunion.

Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases 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 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, 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.

A wealth of new data about the 3D organization of alphavirus particles was obtained in the last few years. This includes the crystal structures of the envelope glycoprotein complexes at neutral and at acid pH, as well as electron microscopy reconstructions of intact virions at neutral pH to resolutions between 7Å and 4Å. The combination has provided unprecedented detail in the description of the alphavirus virion. This review surveys the main features discovered and the implications for the biology of the virus, in particular for the process of disassembly of the glycoprotein shell during entry. The major outstanding questions in this area are also identified and discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Little is known about the three-dimensional organization of rubella virus, which causes a relatively mild measles-like disease in children but leads to serious congenital health problems when contracted in utero. Although rubella virus belongs to the same family as the mosquito-borne alphaviruses, in many respects it is more similar to other aerosol-transmitted human viruses such as the agents of measles and mumps. Although the use of the triple MMR (measles, mumps and rubella) live vaccine has limited its incidence in western countries, congenital rubella syndrome remains an important health problem in the developing world. Here we report the 1.8 Å resolution crystal structure of envelope glycoprotein E1, the main antigen and sole target of neutralizing antibodies against rubella virus. E1 is the main player during entry into target cells owing to its receptor-binding and membrane-fusion functions. The structure reveals the epitope and the neutralization mechanism of an important category of protecting antibodies against rubella infection. It also shows that rubella virus E1 is a class II fusion protein, which had hitherto only been structurally characterized for the arthropod-borne alphaviruses and flaviviruses. In addition, rubella virus E1 has an extensive membrane-fusion surface that includes a metal site, reminiscent of the T-cell immunoglobulin and mucin family of cellular proteins that bind phosphatidylserine lipids at the plasma membrane of cells undergoing apoptosis. Such features have not been seen in any fusion protein crystallized so far. Structural comparisons show that the class II fusion proteins from alphaviruses and flaviviruses, despite belonging to different virus families, are closer to each other than they are to rubella virus E1. This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution. By contrast, in the absence of this constraint, the strictly human rubella virus seems to have drifted considerably into a unique niche as sole member of the Rubivirus genus.

Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation.

A number of viruses transport their genomic material from cell to cell enclosed within a lipid bilayer that is in turn encased within a symmetric protein shell. This review focuses in a group of RNA viruses that have this type of virions. This group includes several of important human pathogenic viruses, such as the hepatitis C virus, dengue virus, chikungunya virus, rubella virus and the bunyaviruses. The best studied are the flaviviruses and the alphaviruses, which have a β-sheet rich class II viral fusion protein used for entry into susceptible cells. We extend here the class II concept to encompass symmetric viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. The first glycoprotein acts as chaperone for the folding of the second one, which carries the membrane fusion function. Since the bunyaviruses, included here, are very similar to the class I arenaviruses in other respects, this analysis highlights the patchwork nature of the various viral functional modules acting at different stages of the virus cycle, which appear assembled from genes of different origins., (© 2011 Blackwell Publishing Ltd.)

Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended "flap" masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process.

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.

Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.

Transport of DNA into preformed procapsids is a general strategy for genome packing inside virus particles. In most viruses, this task is accomplished by a complex of the viral packaging ATPase with the portal protein assembled at a specialized vertex of the procapsid. Such molecular motor translocates DNA through the central tunnel of the portal protein. A central question to understand this mechanism is whether the portal is a mere conduit for DNA or whether it participates actively on DNA translocation. The most constricted part of the bacteriophage SPP1 portal tunnel is formed by twelve loops, each contributed from one individual subunit. The position of each loop is stabilized by interactions with helix alpha-5, which extends into the portal putative ATPase docking interface. Here, we have engineered intersubunit disulfide bridges between alpha-5s of adjacent portal ring subunits. Such covalent constraint blocked DNA packaging, whereas reduction of the disulfide bridges restored normal packaging activity. DNA exit through the portal in SPP1 virions was unaffected. The data demonstrate that mobility between alpha-5 helices is essential for the mechanism of viral DNA translocation. We propose that the alpha-5 structural rearrangements serve to coordinate ATPase activity with the positions of portal tunnel loops relative to the DNA double helix.

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.

Background: A 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 Findings: We 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., Conclusions: The 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.

Semliki Forest virus (SFV) is enveloped by a lipid bilayer enclosed within a glycoprotein cage made by glycoproteins E1 and E2. E1 is responsible for inducing membrane fusion, triggered by exposure to the acidic environment of the endosomes. Acidic pH induces E1/E2 dissociation, allowing E1 to interact with the target membrane, and, at the same time, to rearrange into E1 homotrimers that drive the membrane fusion reaction. We previously reported a preliminary Calpha trace of the monomeric E1 glycoprotein ectodomain and its organization on the virus particle. We also reported the 3.3 A structure of the trimeric, fusogenic conformation of E1. Here, we report the crystal structure of monomeric E1 refined to 3 A resolution and describe the amino acids involved in contacts in the virion. These results identify the major determinants for the E1/E2 icosahedral shell formation and open the way to rational mutagenesis approaches to shed light on SFV assembly.

The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

Fusion of biological membranes is mediated by specific lipid-interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low-pH-induced trimeric form. E1 adopts a folded-back conformation that, in the final post-fusion form of the full-length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple-like deformations in the viral and target membranes, leading to formation of the fusion pore.

The lyssavirus phosphoprotein P is a co-factor of the viral RNA polymerase and plays a central role in virus transcription and replication. It has been shown previously that P interacts with the dynein light chain LC8, which is involved in minus end-directed movement of organelles along microtubules. Co-immunoprecipitation experiments and the two-hybrid system were used to map the LC8-binding site to the sequence (139)RSSEDKSTQTTGR(151). Site-directed mutagenesis of residues D(143) and Q(147) to an A residue abolished binding to LC8. The P-LC8 association is not required for virus transcription, since the double mutant was not affected in its transcription ability in a minigenome assay. Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase, a model for the complex between the peptide spanning residues 140-150 of P and LC8 is proposed. This model suggests that P binds LC8 in a manner similar to other LC8 cellular partners.

Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.

On entering the host cell the rotavirus virion loses its outer shell to become a double-layered particle (DLP). The DLP then transcribes the 11 segments of its dsRNA genome using its own transcriptase complex, and the mature mRNA emerges along the 5-fold axis. In order to better understand the transcription mechanism and the role of VP6 in transcription we have studied three monoclonal antibodies against VP6: RV-238 which inhibits the transcriptase activity of the DLP; and RV-133 and RV-138 which have no effect on transcription. The structures obtained by cryo-electron microscopy of the DLP/Fab complexes and by X-ray crystallography of the VP6 trimer and the VP6/Fab-238 complex have been combined to give pseudo-atomic structures. Steric hindrance between the Fabs results in limited Fab occupancy. In particular, there are on average only three of a possible five Fabs-238 which point towards the 5-fold axis. Thus, Fabs-238 are not in a position to block the exiting mRNA, nor is there any visible conformational change in VP6 on antibody binding at a resolution of 23 A. However, the epitope of the inhibiting antibody involves two VP6 monomers, whereas, those of the non-inhibiting antibodies have an epitope on only one VP6. Thus, the inhibition of transcription may be a result of inhibition of a possible change in the VP6 conformation associated with the transcription of mRNA., (Copyright 2001 Academic Press.)

Crystals of tetragonal hen egg-white lysozyme were grown using Advanced Protein Crystallization Facility (APCF) apparatus under a microgravity environment (SpaceHab-01 mission) and ground control conditions. Crystals were grown from NaCl as a crystallizing agent at pH 4.3. The X-ray diffraction patterns of the best diffracting ground- and space-grown crystals were recorded using synchrotron radiation and an image plate on the W32 beamline at LURE. Both ground- and space-grown crystals showed nearly equivalent maximum resolution of 1.3-1.4 A. Refinements were carried out with the program X-PLOR with final R values of 18.45 and 18.27% for structures from ground- and space- grown crystals, respectively. The two structures are nearly identical with the root-mean-square difference on all protein atoms being 0.13 A. Some residues of the two refined structures show multiple alternative conformations. Two ions were localized into the electron-density maps of the two structures: one chloride ion at the interface between two symmetry-related molecules and one sodium ion stabilizing the loop Ser60-Leu75. The sodium ion is surrounded by six ligands which form a bipyramid around it at distances of 2.2-2.6 A.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.

The FORME package presented herein is designed for modeling purposes: It allows interactive deformation of the protein backbone. General formalism on transformations is introduced and the operators of stretching inside an "acceptance area" and stretching with end-block invariance (i.e., governed by a translational moving) are described. A discussion is presented on the choice of strategy to achieve an interactive deformation tool. Perspectives about complex transformations are presented.

The new routine SYMCRY of the graphics program MANOSK is described in this paper. It is designed to analyze interactions between molecular structures related by crystalline symmetry. The symetric objects can be described in the same referential, to be manipulated as an entity, or in a referential of their own, to undergo correlative real-time movements (via the dials), given the symmetry constraints. Crystal packing can be observed, and any command of the main software MANOSK is available for the symmetric objects, including storage of the coordinates of symmetrics in the final orientation.

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.

Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in Escherichia coli. A cAMP-independent variant, in which Ala-144 is replaced by Thr (CAP91), is activated by analogues of cAMP, such as adenosine, which do not activate the wild-type CAP. In order to test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of 10 mM adenosine, and X-ray diffraction data were measured to 3.5-A resolution. The difference Fourier map calculated with phases from the CAP91 structure showed significant negative density at the position of the phosphate of cAMP bound in one subunit of the CAP91 dimer. Adenosine was preferentially substituted for cAMP in the subunit in the "closed" conformation, while the cAMP-binding site of the "open" subunit was apparently still occupied by cAMP. The structure was refined by restrained least-squares methods to an R factor of 20.2%. Adenosine is not bound in exactly the same position as cAMP; instead, the 5'-OH of adenosine is in a new position that allows formation of two hydrogen bonds with Ser-83, replacing two of the three interactions of the phosphate of cAMP with Arg-82 and Ser-83.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.