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A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an ‘upright’ to a ‘reversed’ conformation. The reversed structure projects a previously buried ‘foot’ domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure1,2. β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3–5. One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins5–8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia colifolding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10–12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.

For broad protection against infection by viruses such as influenza or HIV, vaccines should elicit antibodies that bind conserved viral epitopes, such as the receptor-binding site (RBS). RBS-directed antibodies have been described for both HIV and influenza virus, and the design of immunogens to elicit them is a goal of vaccine research in both fields. Residues in the RBS of influenza virus hemagglutinin (HA) determine a preference for the avian or human receptor, α-2,3-linked sialic acid and α-2,6-linked sialic acid, respectively. Transmission of an avian-origin virus between humans generally requires one or more mutations in the sequences encoding the influenza virus RBS to change the preferred receptor from avian to human, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference. For example, the X-181 strain of the 2009 new pandemic H1N1 influenza virus, derived from the A/California/07/2009 isolate and used in essentially all vaccines since 2009, has arginine at position 226, a residue known to confer preference for an α-2,3 linkage in H1 subtype viruses; the wild-type A/California/07/2009 isolate, like most circulating human H1N1 viruses, has glutamine at position 226. We describe, from three different individuals, RBS-directed antibodies that recognize the avian-adapted H1 strain in current influenza vaccines but not the circulating new pandemic 2009 virus; Arg226 in the vaccine-strain RBS accounts for the restriction. The polyclonal sera of the three donors also reflect this preference. Therefore, when vaccines produced from strains that are never passaged in avian cells become widely available, they may prove more capable of eliciting RBS-directed, broadly neutralizing antibodies than those produced from egg-adapted viruses, extending the established benefits of current seasonal influenza immunizations.

The heterotetrameric Ndc80 complex establishes connectivity along the principal longitudinal axis of a kinetochore. Its two heterodimeric subcomplexes, each with a globular end and a coiled-coil shaft, connect end-to-end to create a ∼600 Å long rod spanning the gap from centromere-proximal structures to spindle microtubules. Neither subcomplex has a known function on its own, but the heterotetrameric organization and the characteristics of the junction are conserved from yeast to man. We have determined crystal structures of two shortened (“dwarf”) Ndc80 complexes that contain the full tetramer junction and both globular ends. The junction connects two α-helical coiled coils through regions of four-chain and three-chain overlap. The complexity of its structure depends on interactions among conserved amino-acid residues, suggesting a binding site for additional cellular factor(s) not yet identified.

Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

Influenza-virus antigenicity evolves to escape host immune protection. Antibody lineages within individuals evolve in turn to increase affinity and hence potency. Strategies for a “universal” influenza vaccine to elicit lineages that escape this evolutionary arms race and protect against seasonal variation and novel, pandemic viruses will require directing B cell ontogeny to focus the humoral response on conserved epitopes on the viral hemagglutinin (HA). The unmutated common ancestors (UCAs) of six distinct, broadly neutralizing antibody lineages from one individual bind the HA of a virus circulating at the time the participant was born. HAs of viruses circulating more than 5 years later no longer bind the UCAs, but mature antibodies in the lineages bind strains from the entire 18-year lifetime of the participant. The analysis shows how immunological memory shaped the response to subsequent influenza exposures and suggests that early imprinting by a suitable influenza antigen may enhance likelihood of later breadth.

Accurate segregation of genetic material in eukaryotes relies on the kinetochore, a multiprotein complex that connects centromeric DNA with microtubules. In yeast and humans, two proteins—Mif2/CENP-C and Chl4/CNEP-N—interact with specialized centromeric nucleosomes and establish distinct but cross-connecting axes of chromatin-microtubule linkage. Proteins recruited by Chl4/CENP-N include a subset that regulates chromosome transmission fidelity. We show that Chl4 and a conserved member of this subset, Iml3, both from Saccharomyces cerevisiae, form a stable protein complex that interacts with Mif2 and Sgo1. We have determined the structures of an Iml3 homodimer and an Iml3-Chl4 heterodimer, which suggest a mechanism for regulating the assembly of this functional axis of the kinetochore. We propose that at the core centromere, the Chl4-Iml3 complex participates in recruiting factors, such as Sgo1, that influence sister chromatid cohesion and encourage sister kinetochore biorientation.

The Saccharomyces cerevisiaemonopolin complex directs proper chromosome segregation in meiosis I by mediating co-orientation of sister kinetochores on the meiosis I spindle. The monopolin subunits Csm1 and Lrs4 form a V-shaped complex that may directly crosslink sister kinetochores. We report here biochemical characterization of the monopolin complex subunits Mam1 and Hrr25 and of the complete four-protein monopolin complex. By purifying monopolin subcomplexes with different subunit combinations, we have determined the stoichiometry and overall architecture of the full monopolin complex. We have determined the crystal structure of Csm1 bound to a Mam1 fragment, showing how Mam1 wraps around the Csm1 dimer and alters the stoichiometry of kinetochore-protein binding by Csm1. We further show that the kinase activity of Hrr25 is altered by Mam1 binding, and we identify Hrr25 phosphorylation sites on Mam1 that may affect monopolin complex stability and/or kinetochore binding in meiosis.

Many biological and chemical processes proceed through one or more intermediate steps. Statistical analysis of dwell-time distributions from single molecule trajectories enables the study of intermediate steps that are not directly observable. Here, we discuss the application of the randomness parameter and model fitting in determining the number of steps in a stochastic process. Through simulated examples, we show some of the limitations of these techniques. We discuss how shot noise and heterogeneity among the transition rates of individual steps affect how accurately the number of steps can be determined. Finally, we explore dynamic disorder in multistep reactions and show that the phenomenon can obscure the presence of rate-limiting intermediate steps.

At least five recent papers have shown an unexpected antigenic relationship between Zika and dengue viruses, with potential implications for vaccines and therapeutics.

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface.

A symposium to remember the life of Raj Rajashankar and to highlight the many scientific achievements to which he contributed was also an occasion to reflect on the fundamental role of personal interactions in the research enterprise.

Chromosome segregation at mitosis depends critically on the accurate assembly of kinetochores and their stable attachment to microtubules. Analysis of Saccharomyces cerevisiaekinetochores has shown that they are complex structures containing ≥50 protein components. Many of these yeast proteins have orthologs in animal cells, suggesting that key aspects of kinetochore structure have been conserved through evolution, despite the remarkable differences between the 125-base pair centromeres of budding yeast and the Mb centromeres of animal cells. We describe here an analysis of S. cerevisiaeNdc10p, one of the four protein components of the CBF3 complex. CBF3 binds to the CDEIII element of centromeric DNA and initiates kinetochore assembly. Whereas CDEIII binding by Ndc10p requires the other components of CBF3, Ndc10p can bind on its own to CDEII, a region of centromeric DNA with no known binding partners. Ndc10p-CDEII binding involves a dispersed set of sequence-selective and -nonselective contacts over ∼80 base pairs of DNA, suggesting formation of a multimeric structure. CDEII-like sites, active in Ndc10p binding, are also present along chromosome arms. We propose that a polymeric Ndc10p complex formed on CDEII and CDEIII DNA is the foundation for recruiting microtubule attachment proteins to kinetochores. A similar type of polymeric structure on chromosome arms may mediate other chromosome–spindle interactions.

The crystal structure of the NFAT1 Rel homology region (RHR) bound to a pseudo-palindromic DNA site reveals an asymmetric dimer interaction between the RHR-C domains, unrelated to the contact seen in Rel dimers such as NFκB. Binding studies with a form of the NFAT1 RHR defective in the dimer contact show loss of cooperativity and demonstrate that the same interaction is present in solution. The structure we have determined may correspond to a functional NFAT binding mode at palindromic sites of genes induced during the anergic response to weak TCR signaling.

ABSTRACTNuclear magnetic resonance spectroscopy demonstrates that the rhesus rotavirus hemagglutinin specifically binds a-anomeric N-acetylneuraminic acid with a Kdof 1.2 mM. The hemagglutinin requires no additional carbohydrate moieties for binding, does not distinguish 3' from 6' sialyllactose, and has approximately tenfold lower affinity for N-glycolylneuraminic than for N-acetylneuraminic acid. The broad specificity and low affinity of sialic acid binding by the rotavirus hemagglutinin are consistent with this interaction mediating initial cell attachment prior to the interactions that determine host range and cell type specificity.

ABSTRACTRotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of ß-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (ΔN10) and 30 residues from the carboxy terminus (ΔC30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells (“virus-like particles” or VLPs) at low pH, with the exception of HPV16 L1 ΔN10, which assembled into a 12-pentamer, T = 1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.

Rotavirus is a major cause of severe, dehydrating childhood diarrhea. VP7, the rotavirus outer capsid glycoprotein, is a target of protective antibodies and is responsible for the calcium-dependent uncoating of the virus during cell entry. We have purified, characterized, and crystallized recombinant rhesus rotavirus VP7, expressed in insect cells. A critical aspect of the purification is the elution of VP7 from a neutralizing monoclonal antibody column by EDTA. Gel filtration chromatography and equilibrium analytical ultracentrifugation demonstrate that, in the presence of calcium, purified VP7 trimerizes. Trimeric VP7 crystallizes into hexagonal plates. Preliminary X-ray analysis suggests that the crystal packing reproduces the hexagonal component of the icosahedral lattice of VP7 on triple-layered rotavirus particles. These data indicate that the rotavirus outer capsid assembles from calcium-dependent VP7 trimers and that dissociation of these trimers is the biochemical basis for EDTA-induced rotavirus uncoating and loss of VP7 neutralizing epitopes.

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares ∼25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g.CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.

The papillomavirus major late protein, L1, forms the pentameric assembly unit of the viral shell. Recombinant HPV16 L1 pentamers assemble in vitro into capsid-like structures, and truncation of ten N-terminal residues leads to a homogeneous preparation of 12-pentamer, icosahedral particles. X-ray crystallographic analysis of these particles at 3.5 Å resolution shows that L1 closely resembles VP1 from polyomaviruses. Surface loops contain the sites of sequence variation among HPV types and the locations of dominant neutralizing epitopes. The ease with which small virus-like particles may be obtained from L1 expressed in E. colimakes them attractive candidate components of a papillomavirus vaccine. Their crystal structure also provides a starting point for future vaccine design.

The 144-kDa λ2 protein of mammalian reovirus catalyzes a number of enzymatic activities in the capping of reovirus mRNA, including the transfer of GMP from GTP to the 5′ end of the 5′-diphosphorylated nascent transcript. This reaction proceeds through a covalently autoguanylylated λ2-GMP intermediate. The smaller size of RNA capping guanylyltransferases from other organisms suggested that the λ2-associated guanylyltransferase would be only a part of this protein. Limited proteinase K digestion of baculovirus-expressed λ2 was used to generate an amino-terminal Mr42,000 fragment that appears to be both necessary and sufficient for guanylyltransferase activity. Although lysine 226 was identified by previous biochemical studies as the active-site residue that forms a phosphoamide bond with GMP in autoguanylylated λ2, mutation of lysine 226 to alanine caused only a partial reduction in guanylyltransferase activity at the autoguanylylation step. Alanine substitution for other lysines within the amino-terminal region of λ2 identified lysine 190 as necessary for autoguanylylation and lysine 171 as an important contributor to autoguanylylation. A novel active-site motif is proposed for the RNA guanylyltransferases of mammalian reoviruses and otherReoviridaemembers.

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 Å resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 Å by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The β propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.

The trimeric, α-helical coiled-coil core of the HIV-1 gp41 ectodomain is thought to be part of a transient, receptor-triggered intermediate in the refolding of the envelope glycoprotein into a fusion-active conformation. In an effort to discover small organic inhibitors that block gp41 activation, we have generated a biased combinatorial chemical library of non-natural binding elements targeted to the gp41 core. From this library of 61,275 potential ligands, we have identified elements that, when covalently attached to a peptide derived from the gp41 outer-layer α-helix, contribute to the formation of a stable complex with the inner core and to inhibition of gp41-mediated cell fusion.

ABSTRACTVariations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal a-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.

ABSTRACTWe have used phage-displayed peptide libraries to identify novel ligands to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120. Screening of libraries of random 12-mers, 7-mers, and cyclic 9-mers produced two families of gp120 binding peptides. Members of a family with the prototype sequence RINNIPWSEAMM (peptide 12p1) inhibit the interaction between gp120 and both four-domain soluble CD4 (4dCD4) and monoclonal antibody (MAb) 17b, a neutralizing antibody that covers the chemokine receptor binding surface on gp120. Peptide 12p1 inhibits the interaction of 4dCD4 with gp120 from three different HIV strains, implying that it binds to a conserved site on gp120. Members of a second family of peptides, with the prototype sequence TSPYEDWQTYLM (peptide 12p2), bind more weakly to gp120. They do not detectably affect its interaction with 4dCD4, but they enhance its binding to MAb 17b. A common sequence motif in the two peptide families and cross-competition for gp120 binding suggest that they have overlapping contacts. Their divergent effects on the affinity of gp120 for MAb 17b may indicate that their binding stabilizes distinct conformational states of gp120. The functional properties of 12p1 suggest that it might be a useful lead for the development of inhibitors of HIV entry.

The Rel family comprises a group of structurally related, eukaryotic transcription factors. The similarity extends over about 300 amino acid residues, the Rel homology region, which is responsible for DNA binding and dimerization. Two independently determined structures of homodimeric NF-κB p50 bound to DNA show the Rel homology regions and the DNA target sites. The protein consists of two β-barrel domains connected by a short linker. Five loops per monomer contact the DNA. Different half-site spacings in the two structures lead to different relative orientations of N- and C-terminal domains.

The crystal structure of the DNA-binding domain of bacteriophage 434 represser (R1-69) in complex with a 20 base-pair DNA fragment has been determined to 2·5 Å resolution. The DNA fragment contains the sequence of the OR2 operator site, which differs from the previously studied OR1 site at three of the variable six central base-pairs. Comparison of the two structures shows that the overall bent conformation of the DNA backbone as well as the pattern of DNA-protein interactions seen in the OR1/R1-69 complex are maintained in the OR2 complex. However, the conformations of the DNA base-pairs am different in the two structures. In particular, the central base-pairs of OR2/R1-69 structure are more co-planar than in OR1/R1-69, and there are no cross-strand "bifurcated" hydrogen bonds. These results show that binding of the protein causes operator DNA to adopt a particular, well-defined backbone conformation, and they reinforce the notion that the energetic cost of achieving this conformation, most likely different for different sequences, can determine, at least in part, the relative affinity of the repressor for different operator sites.

Comparison of the crystal structure of a complex of the phage 434 Cro protein and a synthetic DNA operator with the complex of the same operator and the 434 repressor DNA-binding domain shows different DNA conformations in the two structures. Binding of the protein determines the precise conformation of the DNA in each case.

We report the crystal structure of an extraviral segment of a retrovirus envelope protein, the Moloney murine leukemia virus (MoMuLV) transmembrane (TM) subunit. This segment, which comprises a region of the MoMuLVTM protein analogous to that contained within the X-ray crystal structure of low-pH converted influenza hemagglutinin, contains a trimeric coiled coil, with a hydrophobic cluster at its base and a strand that packs in an antiparallel orientation against the coiled coil. This structure gives the first high-resolution insight into the retrovirus surface and serves as a model for a wide range of viral fusion proteins; key residues in this structure are conserved among C- and D-type retroviruses and the filovirus ebola.

Analysis of the NF-κB p50 homodimer bound to different DNA sequences shows that the protein can recognize half-site spacings of either three or four base pairs. The protein can maintain most of its DNA contacts by a relative reorientation of its two domains.

The crystal structure of a complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator shows that the protein, very similar in conformation to λ repressor, binds to B-form DNA with the second α-helix of a helix–turn–helix motif lying in the major groove.

The rapid appearance of mutations in circulating human influenza viruses and selection for escape from herd immunity require prediction of likely variants for an annual updating of influenza vaccines. The identification of human antibodies that recognize conserved surfaces on the influenza virus hemagglutinin (HA) has prompted efforts to design immunogens that might selectively elicit such antibodies.

The repressor of phage 434 binds to six operator sites on the phage chromosome1–3. A comparison of the sequences of these 14-base-pair (bp) operator sites3reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the represser–operator complex4shows that at each half-site, the 'recognition α-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper4that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434 (ref. 5). We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.

In the crystal structure of a represser–operator complex (the 434 repressor DNA-binding domain and its 14-base pair (bp) operator), Anderson et al.1elsewhere in this issue identify six positions of likely contact between repressor protein and phosphates of the DNA backbone. At each of these positions, electron densities of protein and DNA merge. Experiments presented here indicate that intact 434 repressor approaches these phosphates very closely when it is bound to DNA in solution. Specifically, when any one of these phosphates is ethylated2, repressor cannot bind to the modified operator. We also identify another position where ethylation has a significant but less dramatic effect on repressor binding, and note that in the structure, repressor closely approaches this phosphate. Our results strongly support the idea that the interactions between protein and the DNA phosphate backbone in the crystallized complex1are the same as those made by intact repressor to operator DNA in solution. In addition, our results suggest that DNA is slightly bent by repressor binding.

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates the function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0 Å resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with an RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5′ nucleotide.

Identification of maturation stages of V3-glycan neutralizing antibodies explains the long duration required for their development.