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The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. Mpro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1′ and P2′ residues that are unique among the SARS-CoV-2 Mpro cleavage sites but conserved among homologous junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1′ or P2′ can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* β-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the “upright” conformation present on fully infectious virions to the “reversed” conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

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. Electron cryomicroscopy and cryotomography studies reveal that rotaviruses attach to a target cell through the outer-layer protein VP4, which—following cleavage—rearranges to enable perforation of the membrane and delivery of the viral genome into the host cell.

We describe cross-reactive human antibodies recognizing influenza B viruses spanning nearly 80 years of antigenic drift. Structures show that they engage the receptor-binding site (RBS) of the viral hemagglutinin with strong similarities to their influenza A counterparts, despite structural differences between the RBS of influenza A and B. Our data show that these antibodies readily cross-react with both influenza B Victoria and Yamagata lineages. We also note that all antibodies are encoded by IGHV3-9/IGK1-33. Future research will provide insight into the prevalence of these antibodies in the human population.

Neutralizing antibodies that recognize the SARS-CoV-2 spike glycoprotein are the principal host defense against viral invasion. Variants of SARS-CoV-2 bear mutations that allow escape from neutralization by many human antibodies, especially those in widely distributed (“public”) classes. Identifying antibodies that neutralize these variants of concern and determining their prevalence are important goals for understanding immune protection. To determine the Delta and Omicron BA.1 variant specificity of B cell repertoires established by an initial Wuhan strain infection, we measured neutralization potencies of 73 antibodies from an unbiased survey of the early memory B cell response. Antibodies recognizing each of three previously defined epitopic regions on the spike receptor binding domain (RBD) varied in neutralization potency and variant-escape resistance. The ACE2 binding surface (“RBD-2”) harbored the binding sites of neutralizing antibodies with the highest potency but with the greatest sensitivity to viral escape; two other epitopic regions on the RBD (“RBD-1” and “RBD-3”) bound antibodies of more modest potency but greater breadth. The structures of several Fab:spike complexes that neutralized all five variants of concern tested, including one Fab each from the RBD-1, -2, and -3 clusters, illustrated the determinants of broad neutralization and showed that B cell repertoires can have specificities that avoid immune escape driven by public antibodies. The structure of the RBD-2 binding, broad neutralizer shows why it retains neutralizing activity for Omicron BA.1, unlike most others in the same public class. Our results correlate with real-world data on vaccine efficacy, which indicate mitigation of disease caused by Omicron BA.1.

Immunization or microbial infection can establish long-term B cell memory not only systemically but also locally. Evidence has suggested that local B cell memory contributes to early local plasmacytic responses after secondary challenge. However, it is unclear whether locality of immunization plays any role in memory B cell participation in recall germinal centers (GCs), which is essential for updating their B cell antigen receptors (BCRs). Using single B cell culture and fate mapping, we have characterized BCR repertoires in recall GCs after boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit the progeny of primary GC B cells to recall GCs more efficiently than do distal boosts. Recall GCs elicited by local boosts contain significantly more B cells with elevated levels of immunoglobulin (Ig) mutation and higher avidity BCRs. This local preference is unaffected by blocking CD40:CD154 interaction to terminate active, GC responses. Local boosts with heterologous antigens elicit secondary GCs with B cell populations enriched for cross-reactivity to the prime and boost antigens; in contrast, cross-reactive GC B cells are rare after distal boosts. Our results suggest that local B cell memory is retained in the form of memory B cells, GC B cells, and GC phenotype B cells that are independent of organized GC structures and that these persistent “primed B cells” contribute to recall GC responses at local sites. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving 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 coli folding 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.

Significance Influenza virus and some other human pathogens evolve to evade herd immunity and, hence, to reinfect previously immune individuals. Individual humoral immune responses also evolve, through somatic mutation and selection in a process known as affinity maturation. We describe a detailed molecular “case history” of this coevolution (to our knowledge, the most thorough such study so far) through structural and biophysical analysis of a lineage of human antibodies directed at the influenza hemagglutinin receptor binding site. We show how affinity maturation in this lineage has maintained adaptability (“breadth” of neutralizing activity) by generating a branched and, hence, diversifying phylogeny. The mechanisms underlying affinity maturation appear to ensure such diversification while also enhancing affinity of the most potent resulting antibodies., Affinity maturation of the B cell antigen receptor (BCR) is a conserved and crucial component of the adaptive immune response. BCR lineages, inferred from paired heavy- and light-chain sequences of rearranged Ig genes from multiple descendants of the same naive B cell precursor (the lineages’ unmutated common ancestor, “UCA”), make it possible to reconstruct the underlying somatic evolutionary history. We present here an extensive structural and biophysical analysis of a lineage of BCRs directed against the receptor binding site (RBS) of subtype H1 influenza virus hemagglutinin (HA). The lineage includes 8 antibodies detected directly by sequencing, 3 in 1 principal branch and 5 in the other. When bound to HA, the heavy-chain third complementarity determining region (HCDR3) fits with an invariant pose into the RBS, but in each of the 2 branches, the rest of the Fab reorients specifically, from its position in the HA-bound UCA, about a hinge at the base of HCDR3. New contacts generated by the reorientation compensate for contacts lost as the H1 HA mutated during the time between the donor’s initial exposure and the vaccination that preceded sampling. Our data indicate that a “pluripotent” naive response differentiated, in each branch, into 1 of its possible alternatives. This property of naive BCRs and persistence of multiple branches of their progeny lineages can offer broader protection from evolving pathogens than can a single, linear pathway of somatic mutation.

Re-entry of memory B cells to recall germinal centers (GCs) is essential for updating their B-cell antigen receptors (BCRs). Using single B-cell culture and fate-mapping, we have characterized BCR repertoires in recall GCs following boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit to recall GCs progeny of primary GC B cells more efficiently than do distal boosts. Recall GCs following local boosts contain significantly more B cells with elevated levels ofIgmutations and higher avidity BCRs. This local preference is unaffected by blockade of CD40:CD154 interaction that terminate active, primary GC responses. Local boosts with heterologous antigens elicit secondary GCs with B-cell populations enriched for cross-reactivity to the priming and boosting antigens; in contrast, cross-reactive GC B cells are rare following distal boosts. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses.One Sentence SummaryThe participation of memory B cells in recall germinal centers depends on whether the boost is local or distal to the priming site.

SUMO homeostasis promotes error-free chromosome segregation. Quan et al. report the structure of a targeting peptide of the Ulp2 desumoylase bound to yeast Ctf3/CENP-I. Disrupting the interaction produces hyper-sumoylated kinetochores, demonstrating the existence of a conserved and dedicated pathway for the regulation of kinetochore sumoylation., The step-by-step process of chromosome segregation defines the stages of the cell cycle. In eukaryotes, signals controlling these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to chromosomal centromeres. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for Ulp2, the nuclear enzyme that removes SUMO chains from modified substrates. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle–regulated processes.

Novel animal influenza viruses emerge, initiate pandemics, and become endemic seasonal variants that have evolved to escape from prevalent herd immunity. These processes often outpace vaccine-elicited protection. Focusing immune responses on conserved epitopes may impart durable immunity. We describe a focused, protective antibody response, abundant in memory and serum repertoires, to a conserved region at the influenza virus hemagglutinin (HA) head interface. Structures of 11 examples, 8 reported here, from seven human donors demonstrate the convergence of responses on a single epitope. The 11 are genetically diverse, with one class having a common, IGκV1-39, light chain. All of the antibodies bind HAs from multiple serotypes. The lack of apparent genetic restriction and potential for elicitation by more than one serotype may explain their abundance. We define the head interface as a major target of broadly protective antibodies with the potential to influence the outcomes of influenza virus infection. IMPORTANCE 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 recent discovery of a widely prevalent antibody response to the conserved interface between two HA "heads" (the globular, receptor-binding domains at the apex of the spike-like trimer) has added a new target for these efforts. We report structures of eight such antibodies, bound with HA heads, and compare them with each other and with three others previously described. Although genetically diverse, they all converge on a common binding site. The analysis here can guide immunogen design for preclinical trials.

Significance Immune memory causes the earliest infection by influenza virus to impart a lasting imprint on an individual’s response to later influenza infections or vaccinations. Until recently, that first exposure was always an infection. In the United States and Europe, many infants and toddlers now receive an influenza vaccine before they experience an infection. Because we have little information about immune imprinting by initial exposure to a vaccine, we have studied, in a nonhuman-primate model, how the mode of initial exposure affects primary and secondary antibody responses. Vaccination with influenza hemaglutinnin (HA) protein and infection with a strain bearing the same HA appear to leave distinct imprints—a difference, if confirmed in humans, relevant for next-generation influenza vaccine design., Immune memory of a first infection with influenza virus establishes a lasting imprint. Recall of that memory dominates the response to later infections or vaccinations by antigenically drifted strains. Early childhood immunization before infection may leave an imprint with different characteristics. We report here a comparison of imprinting by vaccination and infection in a small cohort of nonhuman primates (NHPs). We assayed serum antibody responses for binding with hemaglutinnins (HAs) both from the infecting or immunizing strain (H3 A/Aichi 02/1968) and from strains representing later H3 antigenic clusters (“forward breadth”) and examined the effects of defined HA mutations on serum titers. Initial exposure by infection elicited strong HA-binding and neutralizing serum antibody responses but with little forward breadth; initial vaccination with HA from the same strain elicited a weaker response with little neutralizing activity but considerable breadth of binding, not only for later H3 HAs but also for HA of the 2009 H1 new pandemic virus. Memory imprinted by infection, reflected in the response to two immunizing boosts, was largely restricted (as in humans) to the outward-facing HA surface, the principal region of historical variation. Memory imprinted by immunization showed exposure to more widely distributed epitopes, including sites that have not varied during evolution of the H3 HA but that yield nonneutralizing responses. The mode of initial exposure thus affects both the strength of the response and the breadth of the imprint; design of next-generation vaccines will need to take the differences into account.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity—suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants., Graphical abstract, Unbiased charting of memory B cell receptor-encoded antibodies from COVID-19 convalescent subjects identifies seven major antibody competition groups recognizing epitopic regions with group assignment correlating with cross-CoV-reactivity breadth and neutralization potency. SARS-CoV-2 variants tend to escape antibodies from groups with the most potent neutralizers, but many retain affinity, showing that redundant components of a primary immune response establish durable protection from evolving pathogens.

Accurate chromosome segregation during cell division requires engagement of the kinetochores of sister chromatids with microtubules emanating from opposite poles of the mitotic spindle. In yeast, these “bioriented” metaphase sister chromatids experience tension as the corresponding microtubules (one per sister chromatid) shorten. Spindle-assembly checkpoint signaling appears to cease from a kinetochore under tension, which also stabilizes kinetochore-microtubule attachment in single-kinetochore experiments in vitro. The microtubule polymerase, Stu2, the yeast member of the XMAP215/ch-TOG protein family, associates with kinetochores in cells and contributes to tension-dependent stabilization, both in vitro and in vivo. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that amino-acid residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell-cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function. These findings show that the role of Stu2 in tension sensing depends on its association with kinetochores by binding with Ndc80c.

Novel animal influenza viruses emerge, initiate pandemics and become endemic seasonal variants that have evolved to escape from prevalent herd immunity. These processes often outpace vaccine-elicited protection. Focusing immune responses on conserved epitopes may impart durable immunity. We describe a focused, protective antibody response, abundant in memory and serum repertoires, to a conserved region at the influenza hemagglutinin head interface. Structures of eleven examples, eight reported here, from seven human donors demonstrate the convergence of responses on a single epitope. The eleven are genetically diverse, with one class having a common, IGκV1-39, light chain. All of the antibodies bind HAs from multiple serotypes. The lack of apparent genetic restriction and potential for elicitation by more than one serotype may explain their abundance. We define the head interface as a major target of broadly protective antibodies with the potential to influence the outcomes of influenza infection.

Convergent HIV evolution across species Human immunodeficiency virus (HIV) has a highly diverse envelope protein that it uses to target human cells, and the complexity of the viral envelope has stymied vaccine development. Roark et al. report that the immediate and short-term evolutionary potential of the HIV envelope is constrained because of a number of essential functions, including antibody escape. Consequently, when introduced into humans as HIV or into rhesus macaque monkeys as chimeric simian-human immunodeficiency virus, homologous envelope glycoproteins appear to exhibit conserved patterns of sequence evolution, in some cases eliciting broadly neutralizing antibodies in both hosts. Conserved patterns of envelope variation and homologous B cell responses in humans and monkeys represent examples of convergent evolution that may serve to guide HIV vaccine development. Science , this issue p. eabd2638

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide (“Man9-V3”) for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage (“DH270”), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs—the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen., The V3 region of HIV Env elicits broadly neutralizing antibodies (bnAbs) in patients and represents a potential vaccine antigen. Here, Fera et al. show that the structure of a synthetic V3-glycopeptide closely resembles the conformation in intact HIV Env and identify amino acids in bnAbs that are important for neutralization breadth.

Significance Antigenic variation requires frequent revision of annual influenza vaccines. Next-generation vaccine design strategies aim to elicit a broader immunity by directing the human immune response toward conserved sites on the principal viral surface protein, the hemagglutinin (HA). We describe a group of antibodies that recognize a hitherto unappreciated, conserved site on the HA of H1 subtype influenza viruses. Mutations in that site, which required a change in the H1 component of the 2017 vaccine, had not previously “taken over” among circulating H1 viruses. Our results encourage vaccine design strategies that resurface a protein to focus the immune response on a specific region., Circulating influenza viruses evade neutralization in their human hosts by acquiring escape mutations at epitopes of prevalent antibodies. A goal for next-generation influenza vaccines is to reduce escape likelihood by selectively eliciting antibodies recognizing conserved surfaces on the viral hemagglutinin (HA). The receptor-binding site (RBS) on the HA “head” and a region near the fusion peptide on the HA “stem” are two such sites. We describe here a human antibody clonal lineage, designated CL6649, members of which bind a third conserved site (“lateral patch”) on the side of the H1-subtype, HA head. A crystal structure of HA with bound Fab6649 shows the conserved antibody footprint. The site was invariant in isolates from 1977 (seasonal) to 2012 (pdm2009); antibodies in CL6649 recognize HAs from the entire period. In 2013, human H1 viruses acquired mutations in this epitope that were retained in subsequent seasons, prompting modification of the H1 vaccine component in 2017. The mutations inhibit Fab6649 binding. We infer from the rapid spread of these mutations in circulating H1 influenza viruses that the previously subdominant, conserved lateral patch had become immunodominant for individuals with B-cell memory imprinted by earlier H1 exposure. We suggest that introduction of the pdm2009 H1 virus, to which most of the broadly prevalent, neutralizing antibodies did not bind, conferred a selective advantage in the immune systems of infected hosts to recall of memory B cells that recognized the lateral patch, the principal exposed epitope that did not change when pdm2009 displaced previous seasonal H1 viruses.

Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain variable-domain fragment complexes, by studying a complex we failed to crystallize in pursuing an extended project on the human immune response to influenza vaccines.The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen-antibody interface, although small variation in the twist of the rod-likeHA trimer limited the overall resolution to about 4.5Å.Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results.The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.

The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160)3, retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.

Kinetochore molecular architecture exemplifies "form follows function." The simplifications that generated the one-chromosome:one-microtubule linkage in point-centromere yeast have enabled strategies for systematic structural analysis and high-resolution visualization of many kinetochore components, leading to specific proposals for molecular mechanisms. We describe here some structural features that allow a kinetochore to remain attached to the end of a depolymerizing microtubule (MT) and some characteristics of the connections between substructures that permit very sensitive regulation by differential kinase activities. We emphasize in particular the importance of flexible connections between rod-like structural members and the integration of these members into a compliant cage-like assembly anchored on the MT by a sliding molecular ring.

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.

Non-segmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS-RNA viruses carry all the enzymatic functions required for viral mRNA transcription and replication: RNA polymerization, mRNA capping, cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a pre-initiation conformation. Comparison with the likely elongation state, seen in two partial structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.

Summary Kinetochores mediate chromosome segregation during cell division. They assemble on centromeric nucleosomes and capture spindle microtubules. In budding yeast, a kinetochore links a single nucleosome, containing the histone variant Cse4CENP−A instead of H3, with a single microtubule. Conservation of most kinetochore components from yeast to metazoans suggests that the yeast kinetochore represents a module of the more complex metazoan arrangements. We describe here a streamlined protocol for reconstituting a yeast centromeric nucleosome and a systematic exploration of cryo-grid preparation. These developments allowed us to obtain a high-resolution cryoelectron microscopy reconstruction. As suggested by previous work, fewer base pairs are in tight association with the histone octamer than there are in canonical nucleosomes. Weak binding of the end DNA sequences may contribute to specific recognition by other inner kinetochore components. The centromeric nucleosome structure and the strategies we describe will facilitate studies of many other aspects of kinetochore assembly and chromatin biochemistry.

The HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a “closed” conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain -- the “SOSIP” construct -- has many of the relevant properties; it is also particularly suitable for structure determination. Some single-molecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å. Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM. Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env “spike” and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature.

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture. We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the “double-layer particle” or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRpin situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions (“triple-layer particles” or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA,in vitroas well asin vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

Epitope-focused approaches for selective clonal induction of broadly neutralizing antibodies (bnAbs) inform most current vaccine strategies for influenza virus and other rapidly evolving pathogens. The two conserved epitopes on the influenza hemagglutinin (HA) - the “stem” and the receptor-binding site (RBS) on the “head” - are the focus of the current “universal” influenza vaccine development efforts. Because stem-directed serum bnAbs are much less abundant than head-directed ones, we hypothesized that the HA stem bnAbs may be autoreactive and thus eliminated through the mechanisms of self-tolerance. We compared autoreactivity profiles of a set of stem and head-directed bnAbs. Most of the stem bnAbs we examined bound autoantigens; several showed staining of HEp-2 cells. A smaller proportion of the head-directed bnAbs were polyreactive. Gene usage did not correlate with autoreactivity. We suggest that complex foreign antigens may often have surface patches resembling some host epitope; our results indicate that HA stem epitopes resemble a host epitope more frequently than does the RBS.

Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

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 HIV1–3 and influenza virus4–8, 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, respectively9,10. 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 human9,11,12, but passage of a human-derived vaccine candidate in chicken eggs can select for reversion to avian receptor preference13–15. 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 viruses13,14; 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.

Germinal center (GC) B cells evolve towards increased affinity by a Darwinian process that has been studied primarily in genetically restricted, hapten-specific responses. We explored the population dynamics of genetically diverse GC responses to two complex antigens – Bacillus anthracis protective antigen and influenza hemagglutinin – in which B cells competed both intra- and interclonally for distinct epitopes. Preferred VH rearrangements among antigen-binding, naïve B cells were similarly abundant in early GCs but, unlike responses to haptens, clonal diversity increased in GC B cells as early “winners” were replaced by rarer, high-affinity clones. Despite affinity maturation, inter- and intraclonal avidities varied greatly, and half of GC B cells did not bind the immunogen but nonetheless exhibited biased VH use, V(D)J mutation, and clonal expansion comparable to antigen-binding cells. GC reactions to complex antigens permit a range of specificities and affinities, with potential advantages for broad protection.