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International audience; Fab molecules are used as therapeutic agents, and are invaluable tools in structural biology. We report here a method for production of recombinant Fab in Drosophila S2 cells for use in structural biology. Stably transfected S2 cell lines expressing the Fab were created within weeks. The recombinant Fab was secreted, and after affinity and size exclusion chromatography, 16 mg of pure protein were obtained from a liter of cell culture. The Fab was functional and formed a complex with its cognate antigen as demonstrated by co-precipitation and size exclusion chromatography. Biochemical characterization indicated that the Fab from S2 cells is less extensively glycosylated than the Fab obtained by digestion of antibody produced in hybridoma cells, a feature that may be advantageous for the purposes of crystallogenesis. Taken together, obtaining recombinant Fab from the S2 cells has been a faster and considerably more cost-effective method compared with the enzymatic digestion of the monoclonal antibody.

Herpes simplex virus (HSV) fusion with cells requires the gD, gB, and gH/gL glycoprotein quartet. gD serves as a receptor binding glycoprotein. gB and gH/gL execute fusion in an as-yet-unclear manner. To better understand the role of gH/gL in HSV entry, we produced a soluble version of gH/gL carrying a One-STrEP tag (gH t.st /gL). Previous findings implicated integrins as possible ligands to gH/gL (C. Parry et al., J. Gen. Virol. 86:7-10, 2005). We report that (i) gH t.st /gL bound a number of cells in a dose-dependent manner at concentrations similar to those required for the binding of soluble gB or gD. (ii) gH t.st /gL inhibited HSV entry at the same concentrations required for binding. It also inhibited cell-cell fusion in transfected cells. (iii) The absence of β3 integrin did not prevent the binding of gH t.st /gL to CHO cells and infection inhibition. Conversely, integrin-negative K562 cells did not acquire the ability to bind gH t.st /gL when hyperexpressing αVβ3 integrin. (iv) Constitutive expression of wild-type gH/gL (wt-gH/gL) restricted infection in all of the cell lines tested, a behavior typical of glycoproteins which bind cellular receptors. The extent of restriction broadly paralleled the efficiency of gH/gL transfection. RGD motif mutant gH/gL could not be differentiated from wt-gH with respect to restriction of infection. Cumulatively, the present results provide several lines of evidence that HSV gH/gL interacts with a cell surface cognate protein(s), that this protein is not necessarily an αVβ3 integrin, and that this interaction is required for the process of virus entry/fusion.

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.

RSV in 3D Respiratory syncytial virus (RSV) causes pneumonia and bronchiolitis in infants. RSV is an RNA virus in which the genomic RNA forms part of a nuclease-resistant helical ribonucleoprotein complex. Tawar et al. (p. 1279 ) now use x-ray and electron microscopy data to model the structure of this nucleocapsid complex and show how it can template RNA synthesis. The crystal structure shows RNA wrapped around a decameric ring of nucleocapsid protein. Combining this structure with electron microscopy data gives a model that shows how polymerase might read out the RNA bases without disassembling the nucleocapsid helix.

Tremendous progress has been made in recent years in the acquisition and treatment of electron micrographs of biological samples. These developments now allow retrieving much higher-resolution information from the scrutinized object than was possible until recently. In the case of highly symmetric particles, this approach is providing electron density maps comparable to those obtained by X-ray crystallography at least for large assemblies of equivalent sizes. Such accomplishments were made recently with icosahedral viruses, a bacteriophage (1) and an insect virus (2), and now, as reported in this issue of PNAS (3), this approach has revealed the details of the triple-layered architecture of the medically important rotavirus. This work follows from the recent 3D reconstruction of the double-layered rotavirus subparticles at the same level of resolution (4). There has indeed been steady methodological progress in this field. The first 3D fold of a structural protein of a human virus determined by EM, the hepatitis B virus (HBV) core protein, was reported ≈12 years ago (5, 6). The EM map of the HBV core particles obtained at the time was of sufficient quality to resolve the secondary structure elements of an α-helix-rich HBV core protein. From these pioneering studies, we have now moved to an era in which the quality of the EM maps permits the tracing of entire polypeptide chains for much larger proteins. Although the reported high-resolution 3D reconstructions are still special cases because of their high symmetry, the advances are not necessarily limited to symmetrical particles, but are likely to apply to any rigid macromolecular assembly, provided that enough high-quality images are available to reconstruct the object.

International audience; Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 A X-ray structure of a picobirnavirus-an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties.

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Background Japanese encephalitis virus (JEV) is a member of the genus Flavivirus, family Flaviviridae. Flavivirus NS1 glycoproteins are essential proteins, which exhibit a high degree of sequence homology. The NS1 protein is secreted from infected cells as a soluble hexamer and can induce protective immune response in mice against lethal encephalitis. However, its role in neuroinvasion of JEV remains unknown. Our aim is to identify the role of NS1 protein in JEV neuropathogenesis. In this study, we have produced recombinant JEV NS1 protein in Drosophila S2 cells and have characterized its biochemical, biological and antigenic properties.

International audience; 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.

International audience; The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 A resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.

Background An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection. Methods and Findings Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-α was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes. Conclusions Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito., Author Summary Mosquito-transmitted pathogens are a major challenge to humans due to ever-increasing distribution of the vector worldwide. Dengue virus causes morbidity and mortality, and no anti-viral treatment or vaccine are currently available. The virus is injected into the skin when an infected mosquito probes for blood. Among the skin immunocytes, dendritic cells and macrophages are equipped with pathogen-sensing receptors. Our work has shown that dermal macrophages bind the dengue virus envelope protein. We demonstrate that monocyte-derived dermal macrophages are resistant to infection and present evidence that this is due to sequestration of the virus into fusion-incompetent intracellular vesicles. This identifies skin macrophages as the first innate immune cell potentially capable of protecting the human host from infection by dengue virus shortly after a mosquito bite. These findings have important implications for better understanding the early infection events of dengue virus and of other skin-penetrating pathogens.

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host., Author Summary Chikungunya virus (CHIKV) is a reemerging alphavirus responsible for an unprecedented epidemic in countries of the Indian Ocean region, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The most recent epidemic reemergences were documented in Kinshasa, (50,000 estimated cases in 1999–2000), in Indonesia (2001–2003), the Indian Ocean islands of Mayotte, Mauritius, Réunion, and the Seychelles (270,000 cases in 2005–2006 in La Réunion island), and in India (1.4 to 6.5 million estimated cases in 2006–2007). There is a critical lack of knowledge on the biology of CHIKV. In particular, virtually nothing is known about the interaction of CHIKV (and of most alpahaviruses) with human primary cells. We have studied the replication characteristics and the tropism of clinical CHIKV strains from La Réunion. We designed various assays and reagents to follow viral replication, and we report here that adherent cells (epithelial and endothelial cells, primary fibroblasts), as well as macrophages, are sensitive to infection. In contrast, blood cells did not allow viral replication. We also characterized viral entry pathways and sensitivity to interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host. This paper is the result of a collaborative effort between numerous teams from Institut Pasteur, the Groupe Hospitalier Sud Réunion, and other institutions. Our aim was to establish a task force with multiple and complementary expertise on virology, immunology, and cell biology in order to characterize this enigmatic virus.

International audience; 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.

International audience; Glycoprotein G of the vesicular stomatitis virus triggers membrane fusion via a low pH-induced structural rearrangement. Despite the equilibrium between the pre- and postfusion states, the structure of the prefusion form, determined to 3.0 angstrom resolution, shows that the fusogenic transition entails an extensive structural reorganization of G. Comparison with the structure of the postfusion form suggests a pathway for the conformational change. In the prefusion form, G has the shape of a tripod with the fusion loops exposed, which point toward the viral membrane, and with the antigenic sites located at the distal end of the molecule. A large number of G glycoproteins, perhaps organized as in the crystals, act cooperatively to induce membrane merging.

Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E) mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion., Author Summary The fusion of cellular lipid membranes is an essential process in all forms of life. Such membranes are also part of a specific structural class of viruses—so-called enveloped viruses—that include influenza virus, HIV, severe acute respiratory syndrome coronavirus, Ebola virus, yellow fever virus, and many others. The fusion of the viral with a cellular membrane is a key step in the life cycle of these viruses and allows the delivery of their genetic information into cells. This entry step is controlled by specific proteins at the viral surface that are primed to undergo dramatic structural changes and thus drive membrane fusion. An interference with this process can be a powerful means for inhibiting virus replication and fusion inhibitors have recently become a valuable addition to the armamentarium of anti-HIV treatments. In the present study, we identified an intermediate of the fusion pathway of flaviviruses, which comprise mosquito- and tick-transmitted viruses such as yellow fever, dengue, West Nile, Japanese encephalitis, and tick-borne encephalitis viruses. This work has generated further insights into the mechanism of flavivirus membrane fusion and can thus provide new leads for the development of antiviral agents against these important human pathogens.

Nat. Immunol. 16, 170–177 (2015); published online 15 December 2014; corrected after print 27 February 2015 In the version of this article initially published, the sixth author's surname is spelled incorrectly. The correct spelling is Rouvinski. The error has been corrected in the HTML and PDF versions of the article.

International audience; The vesicular stomatitis virus has an atypical membrane fusion glycoprotein (G) exhibiting a pH-dependent equilibrium between two forms at the virus surface. Membrane fusion is triggered during the transition from the high- to low-pH form. The structure of G in its low-pH form shows the classic hairpin conformation observed in all other fusion proteins in their postfusion conformation, in spite of a novel fold combining features of fusion proteins from classes I and II. The structure provides a framework for understanding the reversibility of the G conformational change. Unexpectedly, G is homologous to gB of herpesviruses, which raises important questions on viral evolution.

Key Points Enveloped animal viruses deliver their genetic contents into host cells by a fusion reaction between the virus membrane, which is derived from the host-cell membrane during virus budding, and the host-cell membrane. Studying the molecular mechanisms of virus membrane-fusion reactions is important, as they are paradigms for cellular membrane-fusion reactions and potential targets for antiviral therapies.The fusion reactions are driven by virus membrane-fusion proteins, which undergo a major conformational change that is triggered by interactions with the target cell. Currently, two classes of virus membrane-fusion proteins are known — class I and class II. Class I proteins have been well characterized and refold to a hairpin conformation that drives membrane fusion.The class II membrane-fusion proteins are considered in detail, using the E1 protein of the alphavirus Semliki Forest virus (SFV) and the E protein of the flavivirus tick-borne encephalitis virus (TBE) as examples.In spite of the lack of any detectable amino-acid-sequence similarity, the ectodomains of the alphavirus (E1) and flavivirus (E) fusion proteins have remarkably similar secondary and tertiary structures. Both proteins fold co-translationally with a companion protein, p62 and prM, respectively. One important difference between the viruses is that different budding sites are used — new alphavirus virions bud from the plasma membrane, whereas flavivirus particles bud into the endoplasmic reticulum as immature virions, which are then transported via the exocytic pathway.The structure of the E1 and E proteins is considered in detail, as are the conformational changes that occur during target-membrane insertion and fusion. Unlike class I fusion proteins, which are already in trimeric form before fusion, class II proteins are dimers that must rearrange during fusion to form a stable membrane-inserted homotrimer. However, despite the fact that class I and class II proteins have very different structures, both classes refold during fusion to give a similar overall 'hairpin' conformation.Evidence suggests that class II trimers interact cooperatively during membrane insertion and fusion. A model for five-fold interactions at the fusion site, including the formation of a transient hemifusion intermediate, is proposed.It is likely that class I and II fusion proteins use the same overall mechanism, suggesting that there could be a universal mechanism of membrane fusion. The possibility that there could be further classes of membrane-fusion proteins in addition to class I and class II is discussed., Despite markedly different structures, both class I and class II viral membrane-fusion proteins adopt a hairpin conformation, inducing fusion of viral and cellular membranes. This review focuses on the class II proteins, using Semliki Forest virus and tick-borne encephalitis virus fusion proteins as examples., Structure–function studies have defined two classes of viral membrane-fusion proteins that have radically different architectures but adopt a similar overall 'hairpin' conformation to induce fusion of the viral and cellular membranes and therefore initiate infection. In both classes, the hairpin conformation is achieved after a conformational change is triggered by interaction with the target cell. This review will focus in particular on the properties of the more recently described class II proteins.

International audience; Dengue virus ( DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.

HERV-FRD is a human endogenous retrovirus that entered the human genome 40 million years ago. Its envelope gene, syncytin-2, was diverted by an ancestral host most probably because of its fusogenic property, for a role in placenta morphogenesis. It was maintained in a functional state in all primate branches as a bona fide cellular gene, submitted to a very low mutation rate as compared to infectious retrovirus genomes. The structure of the syncytin-2 protein thus provides a good insight into that of the oldest mammalian retroviral envelope. Here, we report the crystal structure of a central fragment of its "fossil" ectodomain, allowing a remarkable superposition with the structures of the corresponding domains of present-day infectious retroviruses, in spite of a more than 60% divergent sequence. These results suggest the existence of a unique structural solution selected by these proteins for their fusogenic function.

International audience; Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. Here we report the 1.7-angstrom-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature-and mature-like particle assembly in vitro. The peptide inserts as an amphipathic alpha-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact five-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy.

Dengue virus has a highly ordered structure when grown in mosquito cells at 28 °C. The finding that the virus expands into a less ordered form at 37 °C indicates that the human immune system does not see it as we previously thought.

The interaction of a dimeric membrane anchor-free form of the envelope protein E (sE dimer) from tick-borne encephalitis virus with liposomes at acidic pH levels leads to its conversion into membrane-inserted sE trimers. Electron microscopy shows that these trimers have their long dimensions along the threefold molecular axis, which is oriented perpendicularly to the plane of the membrane, where the protein inserts via the internal fusion peptide. Liposomes containing sE at their surface display paracrystalline arrays of protein in a closely packing arrangement in which each trimer is surrounded by six others, suggesting cooperativity in the insertion process. sE trimers, solubilized with nonionic detergents, yielded three-dimensional crystals suitable for X-ray diffraction analysis.

International audience; Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.

International audience; Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.

Amajor step in understanding the molecular interactions that lead to entry of enveloped viruses into their target cells is obtaining structural information on the viral surface glycoproteins at the atomic level. In addition to carrying the main antigenic determinants of the virus, these proteins are responsible for the major steps involved in the entry process, which involve receptor recognition and fusion between viral and cellular membranes. A huge step forward in the field was made >20 years ago, when the laboratories of Don Wiley and John Skehel obtained the crystal structure of the influenza virus hemagglutinin (HA) (1), which carries both receptor-binding and membrane fusion functions. Since then, the x-ray structures of relatively few soluble ectodomains of viral glycoproteins have been determined, compared to the exponential increase of protein structures deposited in the Protein Data Bank in the same period. In particular, structural information on viral envelope proteins combining both receptor-binding and membrane fusion functions is very scarce. Over the years, only the structures of the tick-borne encephalitis (TBE) flavivirus major envelope (E) protein (2) and of the influenza C virus HA-esterase glycoprotein (3) were added to the database. In this issue of PNAS, Modis et al. (4) report the crystal structure of the E protein from dengue virus, a mosquito-borne flavivirus responsible for the highest rate of disease and mortality among members of this viral genus. This structure is therefore a very important addition to the current repertoire. Dengue virus is the most important arthropod-borne human pathogen. The incidence of dengue fever epidemics has increased dramatically over the last few decades (5), and it is estimated that up to 100 million cases occur annually. In addition, a severe form of the disease, dengue hemorrhagic fever (DHF), has emerged in the same period causing ≈ 500,000 cases worldwide each year …

Publisher Summary This chapter describes the three-dimensional structure of rotavirus (RV) viral proteins 6 (VP6). The structural studies of VP6 by X-ray crystallography and by electron microscopy have led to a pseudo-atomic model of its helical assemblies and of the viral middle layer. Calculation of the surface electrostatic potential shows that VP6 is an acidic molecule, indicating that repulsion between capsomers is likely to be responsible for the different assemblies obtained at different pH. It also suggests that the inner shell of the viral particle plays an essential role in compensating the negative electrostatic charges of VP6. Protein VP6 plays a central role in the organization of the virion, acting as an adaptor between two distinct viral functions: cell entry (outer layer) and genomic RNA packaging (inner layer). It also seems to perform additional functions related to a possible concerted conformational switch of the viral particle necessary for transcription.

The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH 7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH 2 -terminal sequencing of the proteolytic fragments showed that they all contained the β-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the α-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.

We have investigated the genetic diversity of dengue type-1 (DEN-1) virus in Brazil. The full nucleotide sequences of three DEN-1 virus isolated from DEN fever (DF) and DEN hemorrhagic fever patients in northeastern Brazil in 1997 (BR/97) and one from a DF patient in the south of Brazil in 2001 (BR/01) were compared to that of the reference strain BR/90 obtained in the city of Rio de Janeiro in 1990. Sequence analysis showed that the structural proteins were remarkably conserved between all isolates. A total of 27 amino acid changes occurred throughout the non-structural proteins. Among them, nine amino acid substitutions were specific of BR/97 and BR/01 isolates, indicating that in situ evolution of these strains had occurred. Within the BR/97 and BR/01 samples, some amino acid substitutions have been previously identified in DEN-1 virus strains sequenced so far, suggesting that recombination events might have occurred.

Rotavirus has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.

In the absence of a method to ensure that crystals can be obtained for any given protein, the possibility of developing scaffolds for protein crystallisation becomes attractive. Among several approaches that could yield scaffolds, two are particularly promising: the first is based on immunoglobulin Fab fragments and immunoglobulin binding proteins while the second is based on fusion proteins. In the Fab based scaffold, the protein of interest is the antigen recognised by the antibody. In the second case, it is a protein fused to one of the scaffold components. The operational difference between the two methods is the existence of a flexible covalent tether compared to a highly specific interaction. The relative merits and disadvantages of each approach are discussed here. We also describe a lattice obtained through a combinatorial approach which appears to have the required properties to be considered a scaffold. The system makes use of an Fab derived from a rheumatoid factor and an Fc-fusion protein. The Fc-fusion system is ideal for enhanced expression of glycoproteins in mammalian cells and provides a useful tag for their purification. The molecular replacement shows a mode of binding for this rheumatoid factor that is not competitive with bacterial Fc-binding proteins. Hence it may be possible to generalise the method to include bacterial expression of fusion proteins with either protein A or protein G as the fusion partner.

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 Å away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-Å resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage φ6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the φ6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.

Class II proteins are viral membrane fusogenic molecules folded essentially as β-sheet and having an internal fusion peptide. In particular, they lack the characteristic central alpha-helical coiled coil present in the post-fusion conformation of all other viral fusion proteins. The regular, icosahedrally symmetric enveloped viruses that have been studied so far, such as flaviviruses, alphaviruses and phleboviruses have been shown to have class II fusion proteins, which in their pre-fusion conformation make an icosahedral shell surrounding the viral membrane. Yet despite having very similar envelope proteins, these viruses belong to three different viral families with totally different genome replication machineries. We have recently identified the rubella virus fusion a belonging to class II, although the virus particles appear pleomorphic and lack icosahedral symmetry. In spite of the lack of any detectable sequence conservation, the available structures indicate that class II proteins have undergone divergent evolution from a distal, ancestral gene. We have now discovered that the cellular fusion protein EFF-1, involved in syncytium formation during the genesis of the skin in nematodes (C. elegans) and in other multicellular organisms, is also folded as a class II viral fusion protein, thereby indicating common ancestry, highlighting an unprecedented amount of exchange of genetic information between viruses and cells. My talk will discuss the implications of this finding, which highlights the intricate exchange of genetic information that has taken place between viruses and cells during evolution. This analysis also suggests a mechanism for the homotypic cell-cell fusion process, which has not been studied so far.

The tick-borne encephalitis (TBE) flavivirus contains two transmembrane proteins, E and M. Coexpression of E and the M precursor (prM) leads to secretion of recombinant subviral particles (RSPs). In the most common form of these RSPs, analyzed at a 19 A resolution by cryo-electron microscopy (cryo-EM), 60 copies of E pack as dimers in a T = 1 icosahedral surface lattice (outer diameter, 315 A). Fitting the high-resolution structure of a soluble E fragment into the RSP density defines interaction sites between E dimers, positions M relative to E, and allows assignment of transmembrane regions of E and M. Lateral interactions among the glycoproteins stabilize this capsidless particle; similar interactions probably contribute to assembly of virions. The structure suggests a picture for trimer association under fusion-inducing conditions.

Rotaviruses are important human pathogens with a triple-layered icosahedral capsid. The major capsid protein VP6 is shown here to self-assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (3.0) or by a high concentration (100 mM) of divalent cations (Ca(2+) and Zn(2+)). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 fits the atomic model determined by X-ray crystallography remarkably well. The 3-fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.

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.

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-Å resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the “fingertips,” that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669–1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species. This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 64.4 Å. During protein export, one of the two oligosaccharides of NS1 is processed into an endo-β- N -acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell.

Insect viruses that cause polyhedrosis produce infectious microcrystals within a cell. These inclusions were used in a study that pushed the state of the crystallographic art to explain their exceptional stability.

We were astonished by the recent letter by R. A. M. Fouchier et al. (“Gain-offunction experiments on H7N9,” 9 August, p. [612][1]; published online 7 August) and the simultaneous publication in Nature (1). We find it odd that an H7N9 influenza A research manifesto signed by a number of flu

The high mutation rate of hepatitis C virus allows it to rapidly evade the humoral immune response. However, certain epitopes in the envelope glycoproteins cannot vary without compromising virus viability. Antibodies targeting these epitopes are resistant to viral escape from neutralization and understanding their binding-mode is important for vaccine design. Human monoclonal antibodies HC84-1 and HC84-27 target conformational epitopes overlapping the CD81 receptor-binding site, formed by segments aa434–446 and aa610–619 within the major HCV glycoprotein E2. No neutralization escape was yet observed for these antibodies. We report here the crystal structures of their Fab fragments in complex with a synthetic peptide comprising aa434–446. The structures show that the peptide adopts an α-helical conformation with the main contact residues F442 and Y443 forming a hydrophobic protrusion. The peptide retained its conformation in both complexes, independently of crystal packing, indicating that it reflects a surface feature of the folded glycoprotein that is exposed similarly on the virion. The same residues of E2 are also involved in interaction with CD81, suggesting that the cellular receptor binds the same surface feature and potential escape mutants critically compromise receptor binding. In summary, our results identify a critical structural motif at the E2 surface, which is essential for virus propagation and therefore represents an ideal candidate for structure-based immunogen design for vaccine development., Author Summary We report here the crystal structures of two neutralization-escape-resistant human monoclonal antibodies in complex with their peptide epitope. Recognition of the hepatitis C virus (HCV) by the humoral immune response is hampered by the high variability of the envelope glycoproteins. However, the contact site analyzed here involves residues that also are believed to interact with the HCV receptor CD81, which the virus cannot mutate without losing viability. The structures reveal a short α-helix in the epitope projecting two hydrophobic residues into a hydrophobic pocket in the paratope, which we propose is similar to the interaction with the receptor. Our results therefore have important implications for vaccine design against this major human pathogen.

Viral Fusion Effectors Viral fusogens are anchored in the viral membrane, and the initial fusogenic conformational change allows them to insert into the cell membrane. This transition is triggered by interactions of the virus particle with the host cell—either binding a cell surface receptor or protons in the acidic endosomal environment. During the fusogenic conformational change, the fusion protein initially adopts an intermediate extended conformation in which a “fusion peptide” projects away from the virus particle and inserts into the target membrane. The fusion protein subsequently collapses into a “hairpin” conformation in which the fusion peptide becomes juxtaposed to the viral transmembrane (TM) segment, thereby juxtaposing the two membranes. The structures of the postfusion hairpin conformation for a number of viruses are presented, highlighting common structural features of the observed hairpins. The overall process of virus-cell fusion and the accompanying conformational changes of the effector proteins have been reviewed, see for example Harrison (2008). The viral fusion proteins analyzed so far fall into three structural classes in which the postfusion form is a trimer of hairpins with different architectures. The predicted location of the fused membrane is outlined above the trimers. The class I motif is characterized by a 7 residue periodicity of nonpolar amino acids (i.e.,“heptad repeats”) that give rise to a central parallel trimeric a-helical coiled coil along the long axis of a rod-shaped molecule (blue in the ribbon diagrams). The N and C termini (labeled in blue and cyan) pack against each other at the membrane proximal end. A “stutter,” a region where the coiled helix unwinds and alters the nonpolar repeat pattern (in red), permits the structural alignment of the various hairpin trimers (Igonet et al., 2011). The heptad repeats are also interrupted by hydrophilic layers (indicated in magenta, with horizontal arrows). Class II proteins are folded essentially as b sheets with no long coiled a helix, and the trimer of hairpins is formed by the parallel interactions of domains I and II (red and yellow). The trimer is further clamped in place by the lateral interactions of domain III (in blue), which stabilizes the hairpin conformation. A stem region (only partially present in the available structures) connects to the TM segment. The fusion peptide is internal, situated in the loop between two b strands, and is therefore termed “fusion loop” (FL). Class III proteins appear to be intermediate between classes I and II: they have a central parallel coiled coil (blue), but they have an elongated, b-sheet-rich fusion domain N terminal to it (yellow) instead of a fusion peptide as in class I. Although this fusion domain is superficially similar to its counterpart in class II proteins, the topological b-strand organization is unrelated, suggesting convergent rather than divergent evolution. The central parallel coiled coil also has a stutter, allowing a close alignment to the class I fusion proteins. The fusion domain of class III proteins has two fusion loops labeled FL1 and FL2. Although there is no amino acid sequence conservation across virus families, the structures show clear homology within classes II and III. However, the simple heptad repeat pattern of class I fusogens is in several cases not sufficient to distinguish between divergent or convergent evolution and therefore, only for a subset of families displayed in the figure do the fusion proteins appear as clearly homologous. Fusion of Intracellular Vesicles A variety of different proteins control the fusion of intracellular vesicles, but the actual fusion effectors are believed to be the SNARE proteins, as reviewed by Sudhof and Rothman (2009). The prefusion form of the SNAREs appears to be largely unstructured, and the postfusion form consists of a hetero-oligomeric complex formed by SNARE subunits that were anchored in the two opposing membranes before their fusion. The SNAREs in their postfusion conformation share with viral proteins of classes I and III an elongated, a-helical, parallel oligomeric coiled-coil organization in which the C-terminal TM segments are juxtaposed at the same end, making a stable protein rod termed “SNAREpin” by analogy to the viral hairpin. The SNAREpin organization differs from the viral proteins because it is a four-stranded coiled coil in which the amino acid sequence of each a helix is different. At the center, there is a hydrophilic, or “ionic,” layer consisting of a central arginine (R) and three glutamine (Q) residues that hydrogen bond the guanidinium group of the arginine. This feature has allowed the classification of the SNAREs into four different categories—R-SNAREs and Qa-, Qb-, and Qc-SNAREs—with one representative of each being necessary for formation of a functional SNAREpin, as displayed in the figure.

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.

The structure of a large fragment of the p50 subunit of the human transcription factor NF-kappa B, bound as a homodimer to DNA, reveals that the Rel-homology region has two beta-barrel domains that grip DNA in the major groove. Both domains contact the DNA backbone. The amino-terminal specificity domain contains a recognition loop that interacts with DNA bases; the carboxy-terminal dimerization domain bears the site of I-kappa B interaction. The folds of these domains are related to immunoglobulin-like modules. The amino-terminal domain also resembles the core domain of p53.

Site-specific substitutions of arginine for lysine in the thermostable D-xylose isomerase (XI) from Actinoplanes missouriensis are shown to impart significant heat stability enhancement in the presence of sugar substrates most probably by interfering with nonenzymatic glycation. The same substitutions are also found to increase beat stability in the absence of any sugar derivatives, where a mechanism based on prevention of glycation can no longer be invoked. This rather conservative substitution is moreover shown to improve thermostability in two other structurally unrelated proteins, human copper, zinc-superoxide dismutase (CuZnSOD) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The stabilizing effect of Lys --> Arg substitutions is rationalized on the basis of a detailed analysis of the crystal structures of wild-type XI and of engineered variants with Lys --> Arg substitution at four distinct locations, residues 253, 309, 319, and 323. Molecular model building analysis of the structures of wild-type and mutant CuZnSOD (K9R) and GAPDH (G281K and G281R) is used to explain the observed stability enhancement in these proteins. In addition to demonstrating that even thermostable proteins can lend themselves to further stability improvement, our findings provide direct evidence that arginine residues are important stabilizing elements in proteins. Moreover, the stabilizing role of electrostatic interactions, particularly between subunits in oligomeric proteins, is documented.

By the use of limited trypsin digestion of purified virions, we generated a membrane anchor-free and crystallizable form of the tick-borne encephalitis virus envelope glycoprotein E. It retained its reactivity with a panel of monoclonal antibodies, and only subtle structural differences from the native protein E were recognized. Treatment with the bifunctional cross-linker dimethylsuberimidate resulted in the formation of a dimer. Crystallization experiments yielded hexagonal rod-shaped crystals suitable for X-ray diffraction analysis.