Biology subjects

Harrison, S. C.

Publications and source records attributed to Harrison, S. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanism of membrane perforation in rotavirus cell entry

Cell entry of non-enveloped, animal viruses requires translocation of a macromolecular assembly across a cellular membrane. Double-stranded RNA viruses introduce into the target cell an inner capsid particle that does not uncoat further. Instead, it extrudes capped viral mRNA by virtue of polymerase and capping activities within it. As described here, we used cryogenic electron tomography to visualize the full course of rhesus rotavirus entry, from cell attachment and virion uptake to release of the subviral particle. The cryo-tomograms and subtomogram averaging of classified subparticles link high-resolution structures of the virion and its components with time series from live-cell fluorescence microscopy. We outlined the mechanism of each step in the entry process, including the membrane perforation step that transfers a subviral particle into the cytosol.

cell biology↗

bioRxiv2025

Structures of folding intermediates on BAM show diverse substrates fold by a uniform mechanism

The outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria contain {beta}-barrel membrane proteins that are assembled by conserved multi-subunit machines. In bacteria, the {beta}-barrel assembly machine (BAM) folds over a hundred compositionally different substrates into barrels that vary greatly in size. Some larger barrels require globular proteins to plug the barrel lumen. How a single machine can assemble such different barrels is unknown. Here we report three structures representing progressively folded stages of a 16-stranded barrel engaged with BAM, as well as the structure of a late-stage folding intermediate of a 26-stranded substrate folding around its soluble lipoprotein plug on BAM. We find that BAM catalyzes folding of these substrates by a uniform mechanism in which BAM undergoes major distortions to accommodate the nascent barrel.

biochemistry↗

bioRxiv2024

Octahedral small virus-like particles of dengue virus type 2

Flavivirus envelope (E) and precursor M (prM) proteins, when ectopically expressed, assemble into empty, virus-like particles (VLPs). Cleavage of prM to M and loss of the pr fragment converts the VLPs from immature to mature particles, mimicking a similar maturation of authentic virions. Most of the VLPs obtained by prM-E expression are smaller than virions; early, low-resolution cryo-EM studies suggested a simple, 60-subunit, icosahedral organization. We describe here the cryo-EM structure of immature, small VLPs from dengue virus type 2 and show that they have octahedral rather than icosahedral symmetry. The asymmetric unit of the octahedral particle is an asymmetric trimer of prM-E heterodimers, just as it is on icosahedral immature virions; the full, octahedrally symmetric particle thus has 24 such asymmetric trimers, or 72 prM-E heterodimers in all. Cleavage of prM and release of pr generates ovoid, somewhat irregular, mature particles. Previous work has shown that mature smVLPs have fusion properties identical to those of virions, consistent with local, virion-like clustering of 36 E dimers on their surface. The cryo-EM structure and the properties of these VLPs described here relate directly to on-going efforts to use them as vaccine immunogens. IMPORTANCEEctopic expression of flavivirus envelope (E) and precursor M (prM) proteins leads to formation and secretion of empty, virus-like particles (VLPs), which are candidate, non-infectious, virion-like components of flavivirus vaccines. We show that the immature particles of a major class of VLPs -- "small VLPs" (smVLPs), which have smaller diameter than those of virion size, -- are octahedrally (rather than icosahedrally) symmetric, with the same clustering of prM and E, as asymmetric trimers of prM-E heterodimers, found on immature virions. Cleavage of prM and formation of mature, smVLPs yields somewhat irregular, ovoid particles. Design and characterization of VLPs as vaccine components will need to take these properties into account.

microbiology↗

bioRxiv2023

A communication hub for phosphoregulation of kinetochore-microtubule attachment

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is an 600-[A] long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long, C-terminal extension of Dam1. Results reported here show, both by in vitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1 and a similarly conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

biochemistry↗

bioRxiv2023

The rotavirus VP5/VP8 conformational transition permeabilizes membranes to Ca2+

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both outer-layer proteins from the particle. The other outer-layer protein, VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

microbiology↗

bioRxiv2022

Structures of the Ndc80 complex and its interactions at the yeast kinetochore-microtubule interface

The conserved Ndc80 kinetochore complex, Ndc80c, is the principal link between spindle microtubules and centromere associated proteins during chromosome segregation. We used AlphaFold 2 to obtain structural predictions of the Ndc80 "loop" region and the Ndc80:Nuf2 globular head domains that interact with the Dam1 subunit of the decameric DASH/Dam1 complex (Dam1c). The predictions guided design of constructs that readily yielded crystal structures, essentially congruent with the predicted ones. The Ndc80 "loop" is a stiff, straight -helical "switchback" structure, and flexibility within the long Ndc80c rod occurs instead at a hinge point between the globular head and the loop. Conserved stretches of the Dam1 C terminus bind Ndc80c with a short helix followed by an extended segment such that phosphorylation of Dam1 serines 257, 265, and 292 by the mitotic kinase Ipl1/Aurora B can release this contact during error correction of mis-attached kinetochores. We integrate the structural results presented here into our current molecular model of the kinetochore-microtubule interface. The model illustrates how multiple interactions between Ndc80c, DASH/Dam1c and the microtubule lattice stabilize kinetochore attachments.

biochemistry↗

bioRxiv2022

Infant antibody repertoires during the first two years of influenza vaccination

The first encounter with influenza virus biases later immune responses. This "immune imprinting", formerly from infection within a few years of birth, is in the U.S. now largely from immunization with a quadrivalent, split vaccine (IIV4). In a pilot study of IIV4 imprinting, we characterized, by single-B-cell cultures, NextGen sequencing, and plasma antibody proteomics, the primary antibody responses to influenza in two infants during their first two years of seasonal influenza vaccination. One infant, who received only a single vaccination in Year 1, contracted an influenza B (IBV) infection between the two years, allowing us to compare imprinting by infection and vaccination. That infant had a shift in hemagglutinin (HA)-reactive B-cell specificity from largely influenza A (IAV)-specific in Year 1 to IBV-specific in Year 2, both before and after vaccination. HA-reactive B cells from the other infant maintained a more evenly distributed specificity. In Year 2, class-switched HA-specific B cell IGHV somatic hypermutation (SHM) levels reached average levels seen in adults. The HA-reactive plasma antibody repertoires of both infants comprised a relatively small number of antibody clonotypes, with one or two very abundant clonotypes. Thus, after the Year 2 boost, both infants had overall B cell profiles that resembled those of adult controls. ImportanceInfluenza virus is a moving target for the immune system. Variants emerge that escape protection from antibodies elicited by a previously circulating variant ("antigenic drift"). The immune system usually responds to a drifted influenza virus by mutating existing antibodies rather than by producting entirely new ones. Thus, immune memory of the earliest influenza exposure has a major influence on later responses to infection or vaccination ("immune imprinting"). In the many studies of influenza immunity in adult subjects, imprinting has been from an early infection, since only in the past two decades have infants received influenza immunizations. The work reported in this paper is a pilot study of imprinting in two infants by the flu vaccine, which they received before experiencing an influenza infection. The results suggest that a quadrivalent (four-subtype) vaccine may provide an immune imprint less dominated by one subtype than does a monovalent infection.

immunology↗

bioRxiv2022

Visualizing Molecular Interactions that Determine Assembly of a Bullet-Shaped Vesicular Stomatitis Virus Particle

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with a non-segmented genome, closely related to rabies virus. Both have characteristic bullet-like shapes. We report the structure of intact, infectious VSV particles determined by cryogenic electron microscopy. By compensating for polymorphism among viral particles with computational classification, we obtained a reconstruction of the shaft ("trunk") at 3.5 [A] resolution, with lower resolution for the rounded tip. The ribonucleoprotein (RNP), genomic RNA complexed with nucleoprotein (N), curls into a dome-like structure with about eight gradually expanding turns before transitioning into the regular helical trunk. Two layers of matrix (M) protein link the RNP with the membrane. Radial inter-layer subunit contacts are fixed within single RNA-N-M1-M2 modules, but flexible lateral and axial interactions allow assembly of polymorphic virions. Together with published structures of recombinant N in various states, our results suggest a mechanism for membrane- coupled self-assembly of VSV and its relatives.

molecular biology↗

bioRxiv2021

Recall of B cell memory depends on relative locations of prime and boost

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 of Ig mutations 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.

immunology↗

bioRxiv2021

Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease

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. It is required for infection, it cleaves the viral polyprotein at multiple sites, and it is conserved among coronaviruses and distinct from human proteases. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes substrates and how the peptide sequence can dictate catalytic efficiency by influencing the position of the scissile bond. One peptide, constituting the junction between viral non-structural proteins 8 and 9 (nsp8/9), has P1 and P2 residues that are unique among SARS-CoV-2 cleavage sites but conserved among nsp8/9 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.

biochemistry↗

bioRxiv2021

Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.

immunology↗

bioRxiv2021

The structural basis for Ulp2 recruitment to the kinetochore

The step-by-step process of chromosome segregation defines the stages of the cell division cycle. In eukaryotes, signaling pathways that control these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to the centromere of each chromosome. 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 the SUMO protease, Ulp2. 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.

cell biology↗

bioRxiv2020

Structural Basis of Stu2 Recruitment to Yeast Kinetochores

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.

biochemistry↗

bioRxiv2020

The structural basis for kinetochore stabilization by Cnn1/CENP-T

Chromosome segregation depends on a regulated connection between spindle microtubules and centromeric DNA. The kinetochore, a massive modular protein assembly, mediates this connection and also serves as a signaling hub that integrates and responds to changing cues during the cell cycle. Kinetochore functions evolve as the cell cycle progresses, culminating in the assurance of a persistent chromosome-microtubule connection during anaphase, when sister chromatids must transit into daughter cells uninterrupted. We previously determined the structure of the Ctf19 complex, a group of kinetochore proteins at the centromeric base of the kinetochore. We now present a high-resolution structure of a Ctf19 complex sub-assembly involved in centromere-microtubule contact: the Ctf3 complex bound to the Cnn1-Wip1 heterodimer. The resulting composite model of the Ctf19 complex and live-cell imaging experiments provide a mechanism for Cnn1-Wip1 recruitment to the kinetochore. The mechanism suggests feedback regulation of Ctf19 complex assembly and unanticipated similarities in kinetochore organization between yeast and vertebrates.

cell biology↗

bioRxiv2019

Structure of a rabies virus polymerase complex from electron cryo-microscopy

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 [A] 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.

molecular biology↗

bioRxiv2019

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated and polyadenylated mRNAs and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates 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 [A] 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 the RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5-nucleotide.

molecular biology↗

bioRxiv2019

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

biophysics↗

bioRxiv2019

In situ structure of rotavirus VP1 RNA-dependent RNA polymerase

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 RdRp in 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 vitro as well as in 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.

molecular biology↗