Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15–gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Bacteriophages (phages) are viruses that infect bacteria and are considered to be the most abundant entities on Earth. Members of the order Caudovirales are double-stranded DNA (dsDNA) tailed phages (1, 2) that can infect a wide variety of hosts, and they are present in many different environments (3). These viruses possess a tail protein complex that is assembled at one special vertex of their icosahedral capsid, known as the portal vertex (4). Connector or portal proteins create an entry channel to the viral capsid and serve as a docking point for the tail complex (4, 5). Tailed phages generally show a common infection strategy and thus have strong structural homologies, although the protein machinery responsible for host adsorption has specific characteristics depending on each phage family (3). Some phages of the Podoviridae family present a short, noncontractile tail that cannot traverse the complex bacterial wall of gram-negative bacteria (6), whereas others do not present tail machinery at all. These latter viruses have developed an alternative mechanism to cross the bacterial envelope using internal capsid proteins or membranes that are able to assemble tubular structures during infection (7–11).Bacteriophage T7 is a well characterized member of the Podoviridae family that infects Escherichia coli. The viral particle is composed of a 55-nm icosahedral capsid and a 23-nm short noncontractile tail (12). The most remarkable structure of the T7 viral particle is the internal core, a cylindrical structure that is ∼290-Å long and ∼170-Å wide located on top of the connector, which stabilizes the packaged DNA inside the capsid (13). This complex is made up of three proteins: gp14 (20.8 kDa), gp15 (84.2 kDa), and gp16 (144 kDa) (9, 13). These internal core proteins are not essential for morphogenesis of the viral capsid, but they are required to translocate the viral genome during the T7 infection process (11, 14, 15). A lytic transglycosylase motif present in gp16 is essential during infection at temperatures below 20 °C to overcome the highly cross-linked peptidoglycan (16–18).As is the case for most phages, T7 first binds reversibly to a primary receptor that allows correct tail orientation in relation to the bacterial surface. Then, an irreversible interaction takes place with the rough lipopolysaccharide (LPS) (19), which causes conformational changes in the tail leading to the opening of the channel. Later, a tubular conduit is assembled, probably composed of the core proteins, and crosses the bacterial wall (9, 11), although it is not clear how this is accomplished. One hypothesis was proposed by Hu et al. (11) in a study using cryo-electron tomography, in which they described the presence of transient tubular structures during T7 infection. They proposed that the core complex formed by gp14, gp15, and gp16 could disassemble after adsorption and pass through the open tail channel in a completely or partially unfolded state (11). According to this hypothesis, partially unfolded gp14 would be ejected through the channel of the tail complex and then refolded to form a pore in the outer membrane of E. coli, which allows unfolded gp15 and gp16 to cross (11, 18). Once in the periplasm, gp15 and gp16 oligomerize as a tubular structure that spans the periplasm and internal membrane and reaches the cytoplasm. When the channel formed by gp14, gp15, and gp16 is complete, translocation of the T7 genome into the bacteria cytoplasm can take place.Here, we report two cryo-electron microscopy (cryo-EM) structures of the T7 core assemblies: gp15 alone (505 kDa) and that of the complex formed by gp15 and gp16 (gp15–gp16 complex; 1.365 MDa). The gp15 protein alone forms a tubular structure in vitro, although its carboxyl-terminal half is disordered. The gp15–gp16 complex is also a tubular structure, but this time with a fully folded gp15. Although only a small fragment of gp16 is observed in the structure of the complex, the solved model comprises the transglycosylase domain. In this article, we have shown that these bacteriophage proteins, which form part of the mature virus in the fully structured core complex with eightfold symmetry (13, 15, 20, 21), are able to unfold during the infection process, exit the phage, and reassemble into a hexameric tubular structure whose size is compatible with the translocation of viral DNA across the bacterial envelope.     

Structural rearrangements in the phage head-to-tail interface during assembly and infection

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal–phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein–DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein–protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.The dsDNA bacterial viruses (phages or bacteriophages) and herpes viruses keep their genetic information packed at high pressure inside an icosahedral protein capsid. During virus particle assembly the genome is translocated into a prebuilt procapsid through a specialized portal vertex of the capsid (1, 2). Termination of the DNA packaging reaction is coordinated with closure of the portal system to avoid leakage of the viral genome. The outflow of DNA is prevented by conformational changes in the portal protein and binding of head completion proteins building the viral genome gatekeeper (3). In bacteriophages, the resultant complex [connector (4)] provides the connection point for the tail. The head-to-tail interface (HTI), or neck, is composed of the connector and of the tail-completion protein(s) found between the connector and the helical tail tube (Fig. S1A) (3, 5). Phage tails are responsible for host cell recognition and delivery of the viral genome to the host cytoplasm (6). At the beginning of viral infection the phage adsorption apparatus, located at the tail end distal from the capsid, binds to the host receptor, generating a signal that triggers opening of the neck (7). DNA then moves through the tail tube to enter the host cell. That tailed bacteriophages are the most abundant biological entities on Earth indicates the evolutionary advantage of this strategy for infecting bacterial cells. Infection by these viruses plays a central role in microbial ecosystems dynamics and in the horizontal transmission of genetic information within the bacterial world (8).Bacillus subtilis tailed bacteriophage SPP1 is a paradigm for viruses with a portal system (9). The viral particle is composed of an isometric icosahedral capsid ∼60 nm in diameter, shielding the 45.9-kbp-long viral chromosome (10). The portal protein gp6 (57.3 kDa subunit mass) is incorporated at a single vertex of the procapsid as a circular oligomer with a central channel that serves as a conduit for DNA passage (11). The portal vertex acts as a platform for the assembly of the viral DNA-translocating motor (12). Termination of DNA packaging is coordinated with disassembly of the motor and binding of gp15 subunits (11.6 kDa) to gp6, extending the portal channel that is closed underneath by the gp16 protein (12.5 kDa) (Fig. S1A) (13). The assembled complex represents the 180-Å-high connector that consists of three stacked cyclical homo-oligomers, each composed of 12 subunits of the portal protein gp6, of the adaptor gp15, and of the stopper gp16 (4, 13). Gp16 operates as a docking platform for the SPP1 preassembled tail tapered by the tail-to-head joining protein gp17 (15 kDa) (14, 15). Binding of the flexible 1,600-Å-long helical tail to the connector completes the formation of the HTI (7, 16). The capsid-distal region of the tail features an adsorption apparatus. Binding of this apparatus to the host cell receptor YueB (17, 18) triggers a domino-like cascade of conformational changes within the gp17.1/gp17.1* tail tube (7, 16, 19), signaling for opening of the gp16 stopper to initiate delivery of the SPP1 genome to the host cell.We report here subnanometer structures of the SPP1 HTI before and after DNA release obtained by cryoEM and single-particle analysis. The EM structures were used for flexible docking of X-ray and NMR atomic models of protein components of the HTI, allowing the uncovering the network of protein–protein and protein–DNA interactions in the complete HTI. The follow-up structure-driven functional analysis unraveled the allosteric mechanism by which the gatekeeper system assembles to lock DNA inside the virion after the genome-packaging reaction. It also provided experimental evidence supporting a model in which reversible diaphragm-like motion is the mechanism that controls viral genome release from the HTI for delivery to the host cell.     

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41_ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41_ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41_ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated.Trimeric envelope glycoprotein (Env gp) spikes on the HIV type 1 (HIV-1) surface mediate entry of the viral genome into the target cell (1, 2). When spikes interact with their cell-surface receptors, a series of conformational changes within the Env culminates in virus–cell membrane fusion. Neutralizing antibodies (NAbs) against various Env epitopes antagonize these events (2, 3). Hence, Env glycoproteins are a focus of vaccine design programs intended to induce NAbs and thereby prevent HIV-1 transmission (3, 4). Env trimers are composed of three gp120 surface glycoprotein subunits and three gp41 transmembrane glycoproteins, the six subunits all associated via noncovalent interactions (5, 6). A critical event in trimer assembly is proteolytic cleavage of the gp160 precursor into its gp120 and gp41 components, a process essential for HIV-1 entry not least because it liberates the fusion peptide (FP) at the gp41 N terminus (5, 6).Trimer-based vaccine strategies involve expressing soluble, recombinant versions of the virion-associated (i.e., native) spikes. To facilitate production and purification, the membrane-spanning and cytoplasmic domains that anchor spikes to the virion, but that are not NAb targets, are eliminated (7–12). However, the resulting proteins, known as gp140s, are highly unstable and disintegrate into their gp120 and gp41-ectodomain (gp41_ECTO) components, making them useless as immunogens. Two fundamentally different protein-engineering strategies have been used to create gp140s that can be produced and purified without falling apart (3, 4, 7–17). The most common method involves eliminating the cleavage site between gp120 and gp41_ECTO, creating uncleaved gp140s (gp140_UNC) where the two subunits remain covalently linked (7–12). Additional trimerization motifs are often added to the gp41_ECTO C terminus (10–12). Our alternative approach is based on the premise that cleavage is a fundamental feature of Env structure and involves stabilizing fully cleaved gp140s. The critical changes are an appropriately positioned disulfide bond (referred to as “SOS”) to link gp120 to gp41_ECTO covalently, and an Ile/Pro (IP) substitution at residue 559 to strengthen inter-gp41_ECTO interactions (13–17). The resulting cleaved trimers are designated SOSIP gp140s (14). Additional modifications have improved their stability, homogeneity, and antigenicity (15–17). Our current design, based on the BG505 subtype A env gene, yields SOSIP.664 trimers that mimic native, virion-associated Env spikes antigenically and when viewed by negative-stain electron microscopy (EM) (17–19).Here we show that cleavage is essential for producing stable, soluble gp140 trimers that resemble native Env spikes. EM studies reveal that purified, trimeric gp140_UNC proteins are heterogeneous and that the irregularly shaped images rarely resemble a native spike; we refer to them as “aberrant configurations” (ACs). In contrast, cleaved SOSIP gp140 trimers are homogeneous and mimic native spikes; we designate them native-like (NL) trimers. The antigenic properties of the cleaved (NL) and uncleaved (AC) trimers, assessed by surface plasmon resonance (SPR) and enzyme-linked immunoabsorbance assays (ELISA), are consistent with the EM images. Nonneutralizing gp120 and gp41_ECTO epitopes are exposed on gp140_UNC trimers but occluded on cleaved ones, whereas quaternary structure-dependent epitopes indicative of proper folding are present only on cleaved trimers. Our findings have substantial implications, because uncleaved trimers are being studied structurally and developed as vaccine candidates (3, 9, 10, 12, 20).     

Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA

Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5′-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5′-triphosphate (ATP) at the subunit interface stabilizes the subunit–subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5′-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein–protein and protein–DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein–ssDNA force field and elucidate the molecular basis of replicative helicase translocation.

Helicases are nucleotide triphosphatase (NTPase)-coupled motors that travel along DNA or RNA (1). Helicases play important roles in many physiological processes including genomic DNA replication. Replicative helicases run at the forefront of the replication fork and separate the double-stranded (ds) parental DNA into two single-stranded (ss) daughter strands, which then serve as templates for DNA synthesis (2, 3). Moreover, helicases are organization hubs for DNA replication by physically interacting with DNA polymerases, primases, ssDNA binding proteins, and adaptor proteins. During their operations, replicative helicases encircle one of the daughter strand ssDNA along which they translocate and sterically exclude the other strand to drive strand separation (2, 3). According to their conserved sequence motifs, helicases can be classified into six superfamilies (SF), with SF1 and SF2 monomeric and SF3 to SF6 hexameric (1). Replicative helicases are hexameric and belong to SF3, SF4, and SF6 families. Helicases in bacteria, bacteriophage, and mitochondria belong to the SF4 family along with RecA-like ATPase domains and display 5′–3′ polarity, while archaeal and eukaryotic SF6 helicases and viral SF3 helicase have AAA+ ATPase domains and display 3′–5′ polarity in their translocation. The structures and mechanisms of hexameric helicase translocation have been extensively studied (2, 3). The homo- or heterohexamers assemble into ring or lockwasher shapes with coiled ssDNA within the central channel. One or two DNA binding loops from the six subunits form a staircase that holds the DNA backbone (4–10). Each subunit in SF3 E1 and SF5 Rho helicases binds one nucleotide, while each subunit from the SF4 and SF6 helicases holds two nucleotides. The DNA binding loops take on distinct conformations in SF3 and SF5 helicases to form a staircase along the DNA backbone. In contrast, the DNA binding loops are rigid in SF4 and SF6 helicases. NTPase sites are located at each subunit interface. Biochemical and single-molecule studies have suggested that NTPs are hydrolyzed sequentially within the helicase hexamer and only one NTPase site fires at a time (11–13). Consistent with that idea, gradual conformational changes of the NTPase sites along the hexameric ring are observed in several helicase–DNA structures, suggesting ordered sequential hydrolysis (4, 5, 7, 8). Taken together, a sequential hand-over-hand mechanism has been proposed for hexameric helicases. An NTPase cycle will drive the DNA binding loop or the subunit at one end of DNA to migrate to the other end so as to form new protein–DNA contacts. Sequential movement of the six subunits enables processive translocation along ssDNA. Nevertheless, how the NTPase cycle is coupled to translocation is unknown, and how a subunit or DNA binding loop migrates a long distance to reach the distal DNA end is unclear.Molecular dynamics (MD) simulations can give insights about dynamic molecular processes that are challenging to obtain using purely experimental methods. Because of the large size of the helicase–DNA complex and the lack of proper force fields for protein–DNA complexes, there have been only a handful of attempts to simulate the helicase translocation process. Coarse-grained simulations have been carried out for SF3 E1 helicase, hepatitis C virus helicase, and the multimeric ATPase chaperonin GroEL (14–16). In another study on LTag helicase, Langevin dynamics simulation has been applied to investigate the protein–DNA interaction in SF3 simian virus 40 helicase (17). However, the coarse-grained DNA models employed in these studies lack the physical benchmark of the ssDNA model and the protein–DNA interactions. Recently, an all-atom simulation on SF5 Rho has revealed how the ATPase cycle is coupled to the transitions of the DNA binding loops (18). So far, there have been no simulation analyses on any SF4 and SF6 helicase family members, which are the major replicative helicases for all three domains of life. Moreover, the DNA conformations and the DNA–protein interactions in the SF4 and SF6 helicases are distinct from those for the SF3 and SF5 helicases. Translocation of SF4 and SF6 helicases has been proposed to involve large-scale conformational changes of an entire subunit, which are absent for the SF3 and SF5 helicases (4, 7, 8).The replicative system from bacteriophage T7 provides a model system for studying DNA replication. T7 gp4 encodes a dual functional protein with primase on its N-terminal domain (NTD) and SF4 helicase on its C-terminal domain. The gp4 helicase exists as heptamers and hexamers in the absence of DNA, with the hexameric form being responsible for DNA unwinding and the heptameric form being possibly responsible for DNA loading (19). In vivo, the gp4 helicase can physically interact with gp5 DNA polymerase and gp2.5 ss DNA binding protein (20). At a replication fork, a single gp4 hexamer and multiple gp5 molecules work cooperatively to catalyze parental DNA unwinding and both leading and lagging strand synthesis (21–23), similar to what happens for other replication systems (24, 25). Recent structures of T7 gp4 with an ssDNA substrate show that the gp4 helicase domain forms a lockwasher-shaped hexamer and interacts with A-form-like ssDNA. The two subunits at the two ends of the hexamer are separated by over 20 Å. The terminal subunit of the lockwasher existed in three distinct conformations, at the 5′-end of DNA, at the 3′-end of DNA, or in the middle, which suggests a subunit translocation pathway. Moreover, the structure suggests that the ATPase site at the 5′-end DNA hydrolyzes ATP first, consistent with the sequential model that has been proposed based on biochemical and single-molecular studies (11, 12).In this report we construct a hybrid coarse-grained force field for protein–ssDNA complexes by combining the OpenAWSEM (Associative memory, Water-mediated, Structure and Energy Model) model for protein and a modified Open3SPN2 model of the nucleic acid components (26). Simulations of gp4 helicase translocation with our force field reveal that ATP hydrolysis is the key determinant that enables subunit translocation. Moreover, our simulation results capture several intermediate states and identify transient protein–DNA and protein–protein interactions that facilitate the long-distance subunit translocation. In summary, the transferable force field developed here is able to simulate motor translocation with large-scale movement.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.As part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80–100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large “terminase” proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7, 8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length molecule with a covalently attached “terminal protein” at the ends (9). The large terminase, which is also an ATPase, then attaches to the protruding end of the portal and assembles into an oligomeric motor that translocates the DNA genome into the empty prohead through the ∼3.5-nm-diameter portal channel using energy from ATP hydrolysis (7, 8). After packaging one unit-length viral genome (headful packaging), the motor dissociates from the full head and the neck and tail proteins assemble on the portal to make an infectious virus.Bacteriophage T4 has been an important model for tailed bacteriophages as well as herpes viruses (10, 11). The T4 packaging motor, a pentamer of gp17 (70 kDa) (large terminase protein) assembled on the gp20 portal dodecamer (12) is the fastest (packaging rate up to ∼2,000 bp/s) of the viral packaging motors studied (13). Gp17 possesses all of the basic enzymatic activities necessary for generating a DNA-full head: ATPase, nuclease, and translocase (14, 15). An oligomeric small terminase protein, gp16, that forms 11-mer and 12-mer rings recognizes the viral genome in vivo, although it lacks strict sequence specificity and is dispensable for packaging in vitro (16). Cryo-EM reconstruction of the packaging motor in complex with the capsid portal, which we will call the “packaging machine,” shows a ring of five gp17 molecules assembled on the prohead portal into a pentameric configuration with the translocation groove facing the channel (12). An electrostatic force-driven translocation mechanism was proposed in which gp17 subunits alternate between the “tensed” (compact) and “relaxed” (extended) conformational states that is coupled to translocation of DNA in a piston-like fashion (12).Genetic and biochemical studies of several packaging systems have delineated the mechanisms of genome recognition and DNA cutting (17, 18). Structural studies (12, 16, 19) and single-molecule optical tweezers (3, 13) and fluorescence spectroscopy (20, 21) approaches have been used to dissect the mechanochemical steps of DNA translocation. However, the transient nature of DNA and protein interactions at the initiation stage, which involve insertion of the dsDNA end into the prohead and triggering of translocation, has been a major challenge (22). The dynamics of motor assembly, timescales of motor–DNA–portal interactions, and mechanism of initiation are poorly understood in any system.Here, we report a single-molecule fluorescence assay that allowed us to dissect packaging initiation starting from a dsDNA end, in real time, by the phage T4 DNA packaging machine. We reconstituted a fully functional minimal T4 packaging complex and imaged individual packaging machines in real time by total internal reflection fluorescence microscopy. Each machine carried out successive DNA translocations and the times for motor assembly and packaging initiation were quantified. Using this assay we found that packaging initiations occurred in bursts with long pauses in between. We discovered that packaging initiation shows unusual plasticity. It can occur through multiple pathways: motor assembly on the portal followed by interaction with DNA and, unexpectedly, direct interaction of DNA with the portal followed by recruitment of the motor subunits. Finally, subtle changes in the ATP binding Walker A P-loop residues that lower the rate of ATP hydrolysis lead to severe defects in packaging initiation. These results provided insights into the dynamics of interactions that lead to single-molecule encapsidation of DNA by a viral packaging machine.     

Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure

Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.There seems to be only a limited number of different virus particle architectures (virion-based structural lineages) due to the limited protein-fold space (1–3). Examples of the uncommon architectures are spindle-, bottle-, and droplet-shaped virions, so far found only in archaeal viruses (4). Archaea, organisms forming the third domain of cellular life, are known to thrive in both moderate and extreme environments (5–7). Interestingly, archaeal viruses are morphologically diverse, resembling eukaryotic viruses in this respect (4). Of these morphotypes, the spindle-shaped (also known as lemon-shaped) viruses are the most common ones in archaea-dominated habitats (8–15). It appears that this architecture is unique. However, deeper structural and biochemical analyses are needed to confirm this claim.Lemon-shaped virions are wider in the middle and narrow toward the ends. Three types of such viruses have been described based on the virion appearance: (i) those with one very short tail, (ii) those with one long tail, and (iii) those having two long tails (16–18). However, based on the comparison of the structural proteins, it was recently proposed that all known lemon-shaped viruses could be classified into two evolutionary lineages or viral families: Fuselloviridae, containing type i viruses, and Bicaudaviridae, containing type ii and iii viruses (19). Most of the isolated lemon-shaped viruses belong to the family Fuselloviridae, whose type species is the Sulfolobus spindle-shaped virus 1 (SSV1). Fuselloviruses infect hyperthermophilic crenarchaea and have one short tail with tail fibers (4, 20). The physical properties of the studied spindle-shaped viruses have recently been summarized (21). However, 3D structural understanding of lemon-shaped viruses remains limited.His1 is the only high-salinity lemon-shaped virus isolate, and it infects an extremely halophilic euryarchaeon, Haloarcula hispanica, and morphologically resembles fuselloviruses (22). However, unlike fuselloviruses, which have a circular double-stranded DNA (dsDNA) genome, His1 has a linear dsDNA genome encoding a putative type-B DNA polymerase; consequently, His1 has been classified in the floating genus Salterprovirus (20, 23). The 14,462-bp genome of His1 is predicted to have 35 ORFs, and 4 of these have been shown to encode structural proteins of the virion (21, 23). His1 virion contains one major capsid protein (MCP), VP21. In addition, a few minor ones have been detected: VP11, VP26, and VP27. Interestingly, VP21 exists in two forms. One form is lipid modified, although there is no detectable lipid bilayer in the His1 virion (21). The His1 MCP is 47% similar to the MCP of SSV1, indicating that His1 and fuselloviruses may share a common ancestor, and it has been proposed that His1 could be classified into the family Fuselloviridae (19, 21). In addition to the DNA polymerase, the His1 genome is predicted to encode an ATPase and a glycosyltransferase (21, 23). His1, like the other lemon-shaped viruses, is nonlytic, and many of them also encode an integrase (16, 17, 21, 24–26). Despite its typically hypersaline environment, His1 tolerates a variety of salinities, from 50 mM up to 4 M NaCl (21).Few attempts have been made to determine the structures of nonicosahedral viruses, mainly due to the fact that their pleomorphic nature introduces great challenges in particle structure classification and averaging. Recent advances in image-processing methods in cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) have been demonstrated to be a useful tool in the structural studies of nonicosahedral viruses, such as Tula hantavirus (27) and the immature capsid in HIV-1 virus (28), and some archaeal viruses, such as pleomorphic, two-tailed spindle-shaped, and linear viruses (17, 21, 29) and bottle-shaped and filamentous archaeal viruses (30–32). Recent structural studies of the spindle-shaped virus SSV1 revealed its 3D structure with a spindle body and a short tail at one end (33).Here, we used cryo-ET and symmetry-free and model-free subtomogram averaging to reveal the previously unrecognized tail organization of the lemon-shaped virus His1, with a central tail hub and six surrounding tail spikes. Further analysis of a larger population of subtomograms showed variable dimensions of the lemon particles but a constant structure of the tail. Biochemical analysis of the virion under different chemical conditions revealed unexpected biochemical properties of the His1 virion, which may be relevant to the life cycle of this virus.     

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti–coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4⁺ T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2–V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.The development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic (1). However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate into the host chromosomes, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope in the native spikes that protrude from the virion surface (2). These spikes are composed of homotrimers of heterodimers of the envelope glycoprotein subunits gp120 and gp41 maintained in an energetically unfavorable, metastable conformation (3, 4). Upon binding to CD4 and a coreceptor such as CCR5 or CXCR4, gp120 undergoes dramatic conformational changes that lead to a low-energy state, creating permissive conditions for activation of the gp41 fusogenic mechanism (3). In the prefusion conformation, gp120 effectively conceals its highly conserved receptor- and coreceptor-binding sites from antibody recognition, imposing a high-entropy penalty for interaction with CD4 or antibodies to the coreceptor-binding site such as 17b; in contrast, in the open, low-energy conformation, gp120 interacts with CD4 and 17b with minimal thermodynamic changes (4, 5). This conformational masking of the vulnerable receptor- and coreceptor-binding sites is believed to be a primary mechanism of immune evasion by HIV-1 (4).The inherent conformational flexibility of gp120, along with the extensive N-linked glycosylation that covers most of the exposed surface of the glycoprotein, has severely hampered attempts to elucidate the native structure of the HIV-1 envelope spike. As a consequence, most of the available high-definition structures of gp120 have been obtained with deglycosylated, variable loop-truncated core monomers in complex with stabilizing ligands such as soluble CD4 (sCD4) (6–10). Important information regarding the overall conformation and ligand interactions of the trimeric spike at intermediate resolution has emerged from the use of increasingly refined cryo-electron microscopy (cryo-EM) technologies (11–17). Moreover, the crystal structure of a stabilized, soluble, cleaved gp140 trimer (BG505 SOSIP.664) at 4.7-Å resolution was reported recently (18). However, despite these advances, many critical aspects related to the structural mechanisms of HIV-1 immune vulnerability and evasion remain unresolved. In particular, the fine molecular details of the interaction between the second and third variable loops (V2 and V3, respectively) of gp120, which are believed to play a critical role in stabilizing the prefusion envelope structure (19–21), are elucidated only partially. Functionally, V2 and V3 cooperate in the formation of quaternary epitopes targeted by some of the most potent and broadly neutralizing mAbs hitherto identified (22, 23), and V2 effectively masks neutralization epitopes in V3 (24–27). Cryo-EM studies have provided evidence that in the prefusion conformation V2 and V3 are spatially contiguous and account for most of the density at the apex of the trimeric envelope spike (12–17). Although various fragments of V2 and V3 were crystallized separately using antibody-complexed synthetic peptides (28, 29), scaffolded chimeric constructs of the first and second variable loops (V1V2) (30, 31), or a V3-containing gp120 core monomer (8), the only study in which the two loops were visualized simultaneously is the recent report of the BG505 SOSIP.664 trimer crystal structure (18). In this artificially stabilized trimer, which displays several antigenic features of the native envelope (32), V2 and V3 appear to interact directly at the trimer apex with the V3 β-hairpin extensively buried under the V1V2 four-stranded Greek-key β-sheet (18).In the present study, we provide evidence that the conserved central region of the gp120 V2 loop contains previously unrecognized sulfated tyrosines that, in the CD4-unbound prefusion state, mediate intramolecular interaction between V2 and the CCR5-binding site at the base of V3. Our results suggest that the sulfotyrosine-bolstered interaction between V2 and V3 is a key structural constraint that stabilizes the native conformation of the HIV-1 envelope trimer.     

Tension-dependent stabilization of E-cadherin limits cell–cell contact expansion in zebrafish germ-layer progenitor cells

Tension of the actomyosin cell cortex plays a key role in determining cell–cell contact growth and size. The level of cortical tension outside of the cell–cell contact, when pulling at the contact edge, scales with the total size to which a cell–cell contact can grow [J.-L. Maître et al., Science 338, 253–256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell–cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell–cell contact size is limited by tension-stabilizing E-cadherin–actin complexes at the contact.

For multicellular organisms to form, cells need to establish stable and long-lasting contacts. Consequently, insight into the molecular and cellular mechanisms by which cell–cell contacts are being formed and maintained is central for understanding how multicellularity has emerged in evolution. Adhesion between cells is mediated by various cell–cell adhesion molecules, among which cadherins constitute a key family of adhesion receptors mediating selective Ca²⁺-dependent cell–cell adhesion (1, 2). While much progress has been made in identifying how cadherin adhesion molecules can trigger cell–cell contact formation by binding to each other and associated molecules, such as catenins (3–5), comparably little is known on how cadherins transduce forces between cells and how such force transduction feeds back on the organization and function of cadherins at cell–cell contacts.Cadherins—and in particular, classical cadherins—are thought to function in cell–cell contact formation in three different ways (6, 7). 1) They promote cell–cell contact formation by directly lowering interfacial tension at the cell–cell contact zone (8). How cadherins achieve this is not yet entirely clear, but the generation of lateral pressure through cadherin-mediated molecular crowding at the contact zone has been proposed as one potential effector mechanism (9, 10). 2) Signaling from cadherins modulates the actomyosin cytoskeleton at the contact site, thereby controlling contact growth and maintenance (11). Effector molecules involved in this process include RhoA and Arp2/3, which both are repressed when cadherins bind over the contact, and Rac, which is activated upon cadherin binding (12, 13). 3) Cadherins transduce pulling forces from the contractile actomyosin cortices of the contacting cells over the contact site (6, 14, 15). This force transduction allows the contact to grow and reach steady state after those forces are balanced at the contact. Data on cultured cells and primary cells from zebrafish embryos support a critical function of cadherins in contact expansion. They are thought to disassemble the actomyosin cortex at the contact site and mechanically couple the cortices of the contacting cells at the contact edge (6, 16). These observations led to a model where pulling forces at the contact edge, originating from the contractile cortices of the contacting cells, are transduced by cadherins over the contact and drive contact expansion. Consequently, the size of the contact is expected to scale with the ratio of cortical tension at the cell–cell vs. the cell–medium interface (6, 9).Cadherins at cell–cell contacts not only transduce forces between the contacting cells but are also affected by the forces to which they are subjected. Studies on culture cells have provided evidence that tension at cadherin cell–cell adhesion sites promotes cadherin clustering and reduces their turnover at the contact site (16–18). How tension functions in those processes is not yet fully understood, but tension-induced stabilization of filamentous actin (F-actin) (16, 19, 20) and unfolding of α-catenin (21), a key component of the cadherin adhesion complex (22, 23), are involved. Unfolding of α-catenin is thought to reveal cryptic binding sites to Vinculin (21, 24), which again enhances binding of α-catenin to F-actin by simultaneously binding to both molecules (17, 20, 25, 26).Yet, how mechanosensing of cadherin cell–cell adhesion sites affects the function of cadherins in contact expansion and maintenance remains unclear. To address this question, we tested how changes in cortex tension affect contact expansion of zebrafish primary germ-layer progenitor cells. Contrary to previous expectations (6), we found that above a critical threshold level of tension, the size of cell–cell contacts becomes smaller rather than bigger. We further found that this restricting influence of cortex tension on contact growth is due to high tension promoting cytoskeletal anchoring of E-cadherin, leading to enhanced clustering and stability of E-cadherin at the contact.     

Mammalian sperm hyperactivation regulates navigation via physical boundaries and promotes pseudo-chemotaxis

Mammalian sperm migration within the complex and dynamic environment of the female reproductive tract toward the fertilization site requires navigational mechanisms, through which sperm respond to the tract environment and maintain the appropriate swimming behavior. In the oviduct (fallopian tube), sperm undergo a process called “hyperactivation,” which involves switching from a nearly symmetrical, low-amplitude, and flagellar beating pattern to an asymmetrical, high-amplitude beating pattern that is required for fertilization in vivo. Here, exploring bovine sperm motion in high–aspect ratio microfluidic reservoirs as well as theoretical and computational modeling, we demonstrate that sperm hyperactivation, in response to pharmacological agonists, modulates sperm–sidewall interactions and thus navigation via physical boundaries. Prior to hyperactivation, sperm remained swimming along the sidewalls of the reservoirs; however, once hyperactivation caused the intrinsic curvature of sperm to exceed a critical value, swimming along the sidewalls was reduced. We further studied the effect of noise in the intrinsic curvature near the critical value and found that these nonthermal fluctuations yielded an interesting “Run–Stop” motion on the sidewall. Finally, we observed that hyperactivation produced a “pseudo-chemotaxis” behavior, in that sperm stayed longer within microfluidic chambers containing higher concentrations of hyperactivation agonists.

The navigational mechanisms that regulate sperm migration through the complex and dynamic physical and chemical environments of the female reproductive tract to the site of fertilization are poorly understood (1, 2). Over many years, studies have revealed that the biophysical navigational cues for sperm in the female tract include fluid flow (3–7), wall architecture (8–12), ambient rheological properties such as fluid viscoelasticity (13), and possible temperature gradients (14, 15). There is also evidence that biochemical cues from the female tract serve to modulate sperm migration (16). These may include chemoattractants (17, 18), molecular triggers that change sperm flagellar beating patterns (19–21), and sperm receptors on the epithelium of the tract that anchor sperm (22–24).The in vivo biochemical factors that transform sperm flagellar beating patterns are not precisely known (16), but in vitro exposure to certain chemical stimuli results in similar transformation of the flagellar beating pattern (25). Specifically, exposure to certain chemical stimuli results in the rise of cytoplasmic Ca²⁺ in sperm (26, 27) through either activation of CATSPER membrane ion channels and flux of exogenous Ca²⁺ ions into the flagellum (28, 29) or by mobilization of intracellular Ca²⁺ stores (30–32) or both. In turn, this rise of cytoplasmic Ca²⁺ concentration results in an increase of asymmetry in the flagellar beat cycle. This process is called “hyperactivation,” and it is required for fertilization (33, 34), as it enhances the ability of sperm to penetrate the matrix of the oocyte’s cumulus oophorus and zona pellucida to reach the plasma membrane of the oocyte (21). Furthermore, there is evidence that hyperactivation assists sperm swimming through viscoelastic substances in the female reproductive tract (35) and plays a role in detaching sperm from epithelial cells in the oviduct (36). It has been observed that hyperactivation is stimulated via a concentration-dependent mechanism (21).Although past findings have revealed roles that hyperactivation plays in supporting the success of fertilization, it remains elusive whether this functional state of motility is directly involved in sperm navigation within the female reproductive tract. Accordingly, we hypothesized that hyperactivation influences sperm–sidewall interactions and thus regulates sperm navigation via nearby wall architecture. Furthermore, we expected this regulatory mechanism to be dependent on the concentration of hyperactivation agonists.Here, using microfluidic experimentation with bovine sperm as well as theoretical and computational modeling, we investigated the effect of hyperactivation on hydrodynamic sperm–sidewall interactions in both standard and viscoelastic media. We used two established pharmacological agents to trigger hyperactivation in sperm: caffeine (25) and 4-aminopyridine (4-AP) (37). We demonstrated that hyperactivation directly regulates sperm interactions with sidewalls of our microfluidic reservoirs and thus navigation via physical boundaries. As a result of this concentration-dependent regulatory mechanism, we observed a “pseudo-chemotaxis” phenomenon in which sperm accumulated within reservoirs with higher concentrations of hyperactivation agonists. Our results revealed a potential role of hyperactivation in the navigational response of sperm to biochemical cues within the female reproductive tract.     

miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

It is well known that the nonmalignant stromal components in tumors play pivotal roles in tumor progression and therapeutic responses (1, 2). Macrophages are a major component of tumor microenvironment and display considerable phenotypic plasticity in their effects toward tumor progression (3–5). Classically activated (M1) macrophages often exert direct tumor cytotoxic effects or induce antitumor immune responses by helping present tumor-related antigens (6, 7). In contrast, tumoral cues can polarize macrophages toward alternative activation with immunosuppressive M2 properties (6–8). Numerous studies have firmly established the protumor effects of M2-like tumor-associated macrophages (TAMs) and the association of TAMs with poor prognosis of human cancer (9–11). However, how tumors induce the coordinated molecular and phenotypic changes in TAMs for M2 polarization remains incompletely understood, impeding the designing of TAM-targeting strategies for cancer intervention. In addition, drug delivery also represents a hurdle for therapeutic macrophage reprogramming.Noncoding RNAs, including microRNAs, have been shown to play vital roles in various pathological processes of cancer (12). The microRNA miR-182 has been implicated in various developmental processes and disease conditions (13–15). Particularly, it receives extensive attention in cancer studies. Prevalent chromosomal amplification of miR-182 locus and up-regulation of its expression in tumors have been observed in numerous cancer types including breast cancer, gastric cancer, lung adenocarcinoma, colorectal adenocarcinoma, ovarian carcinoma, and melanoma (16–21). miR-182 expression is also linked to higher risk of metastasis and shorter survival of patients (20, 22–24). Functional studies showed that miR-182 expression in cancer cells plays vital roles in various aspects of cancer malignancy, including tumor proliferation (25–29), migration (30, 31), invasion (16, 32, 33), epithelial-mesenchymal transition (34–36), metastasis (21, 37, 38), stemness (30, 39, 40), and therapy resistance (41, 42). A number of target genes, including FOXO1/3 (18, 21, 43–45), CYLD (46), CADM1 (47), BRCA1 (27, 48), MTSS1 (34), PDK4 (49), and SMAD7 (35), were reported to be suppressed by miR-182 in cancer cells. Our previous work also proved that tumoral miR-182 regulates lipogenesis in lung adenocarcinoma and promotes metastasis of breast cancer (34, 35, 49). Although miR-182 was established as an important regulator of cancer cell malignancy, previous studies were limited, with analyses of miR-182 in cultured cancer cells and transplanted tumors. Thus, the consequences of miR-182 regulation in physiologically relevant tumor models, such as genetically modified mice, have not been shown. More importantly, whether miR-182 also plays a role in tumor microenvironmental cell components is unknown.In this study, we show that miR-182 expression in macrophages can be induced by breast cancer cells and regulates TAM polarization in various tumor models of mice. In addition, miR-182 inhibition with TAM-targeting exosomes demonstrates promising efficacy for cancer treatment.     

On the question of whether lubricants fluidize in stick–slip friction

Intermittent sliding (stick–slip motion) between solids is commonplace (e.g., squeaking hinges), even in the presence of lubricants, and is believed to occur by shear-induced fluidization of the lubricant film (slip), followed by its resolidification (stick). Using a surface force balance, we measure how the thickness of molecularly thin, model lubricant films (octamethylcyclotetrasiloxane) varies in stick–slip sliding between atomically smooth surfaces during the fleeting (ca. 20 ms) individual slip events. Shear fluidization of a film of five to six molecular layers during an individual slip event should result in film dilation of 0.4–0.5 nm, but our results show that, within our resolution of ca. 0.1 nm, slip of the surfaces is not correlated with any dilation of the intersurface gap. This reveals that, unlike what is commonly supposed, slip does not occur by such shear melting, and indicates that other mechanisms, such as intralayer slip within the lubricant film, or at its interface with the confining surfaces, may be the dominant dissipation modes.Intermittent sliding (stick–slip) of solids in contact is an everyday effect, such as in the squeak of hinges or the music of violins, when the bow slides past the strings, or, at a different scale, in earthquakes (where tectonic plates slide past each other). Such solid sliding is a major cause of frictional dissipation, and can persist even in the presence of lubricants (1). At a nanotribological level, surface force balance (SFB) measurements, supported by theory and computer simulations, have shown that when simple organic liquids are confined between atomically smooth, solid (mica) surfaces to films thinner than some six to eight molecular layers, they may become solid-like, and are often layered (2–14). Subsequent sliding of the surfaces across such films when they are subjected to shear may then take place via stick–slip motion (15, 16). During the stick part, the surfaces are in rigid contact until the shear force between them exceeds the static friction, at which point they slip rapidly past each other (relaxing the shear stress) and then stick again, in a repeating cycle. The issue of how the confined (lubricant) layer progressively yields and then becomes rigid again during such stick–slip sliding has been intensely studied over the past several decades, not least because a better understanding may result in improved lubrication approaches.The molecular basis of the stick–slip cycle in sheared solid-like lubricant films as described above is not well understood (17–28). This is at least in part because, experimentally, it is very challenging to capture what happens to the lubricant layer during the fleeting, individual slip events taking place in the nanometrically confined film. Even when measured under controlled conditions, as in the SFB, these slip events are not only of very short duration [ca. 20 ms (18)] but generally occupy only a tiny fraction of the stick–slip cycle, with the surfaces in nonsliding contact (stick) for almost the entire cycle period. For this reason, much of our understanding has been derived from theoretical modeling and computer simulation studies (17, 19–25, 27–29). Classically, these almost all suggest that the stick–slip motion involves periodic shear melting transitions and resolidification of the film as it undergoes transition between solid-like and liquid-like phases during sliding. Even where there is some disagreement in the model details [for example, on the precise mechanism by which the films solidify at the end of the slip (22, 25)], they maintain the essential idea of fluidization of the lubricant layers during the slip part of the stick–slip cycle. In the shear-induced solid to liquid transition (fluidization), a density change is also expected because the fluidized phase is less dense than the solid phase. This leads to a volumetric expansion and contraction cycle (corresponding respectively to slip and stick), with a dilation of the thin lubricant film during the slip event (17, 23, 25, 27). Some more recent simulations suggest that slip may occur at the wall–fluid interfaces or via interlayer slip within the film rather than via film melting (19, 27, 28), although the scenario of lubricant fluidization during slip is the generally accepted mechanism.There have been few experimental studies on individual slips during stick–slip sliding across lubricant films, and none where the film thickness in such fleeting events has been examined (15, 16, 18, 30–32). Clues may also be extracted from stick–slip motion of confined granular systems under shear, where numerical simulations (33, 34) and some experiments (35–37) suggest that fluidization and dilation may play a role in the stick–slip instability. While this is suggestive, differences between granular layers and lubricant films include not only five orders of magnitude between size of grains and of molecules but, in particular, the issue of molecular interactions, negligible in granular shear but all-important when shearing lubricants.In the present study, we examine directly the individual slip events during stick–slip sliding across thin lubricant films, and in particular the issue of film dilation during the fleeting slip motion itself. This is done to provide “smoking gun” evidence concerning the issue of film fluidization, where such dilation is expected to be a clear signature. We confine a thin (few nanometers) model liquid film between smooth solid surfaces in an SFB, shear it, and monitor the film thickness during stick–slip sliding via fast video microscopy. To overcome the major challenge presented by the shortness of the slip events, which occupy only some 1% of the stick–slip cycle over which a subnanometer dilation needs to be detected against a comparable level of noise, we analyze our data using tools from classical signal detection theory to correlate the slip events with the instantaneous value of the film thickness.     

Insect’s intestinal organ for symbiont sorting

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host–symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ’s ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host’s control and symbiont’s motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.Diverse organisms are obligatorily associated with microbial symbionts, which significantly contribute to their adaptation and survival (1–3). In such symbiotic associations, the host organisms often develop specialized cells, tissues, or organs for harboring their specific microbial partners [for example, root nodules in the legume–Rhizobium symbiosis (4, 5), symbiotic light organs in the squid–Vibrio symbiosis (6, 7), and bacteriocytes in the aphid–Buchnera symbiosis (8, 9)].These microbial symbionts are either acquired by newborn hosts from the environment every generation as in the legume–Rhizobium and the squid–Vibrio symbioses or transmitted vertically through host generations as in the aphid–Buchnera symbiosis (10). In the environmentally acquired symbiotic associations, it is essential for the host organisms to recognize and incorporate specific symbiotic bacteria while excluding a myriad of nonsymbiotic environmental microbes (6, 11). In the vertically transmitted symbiotic associations, it is important for the host organisms to selectively transmit their own symbiotic bacteria while excluding parasitic/cheating microbial contaminants (12–14). Hence, it is expected that the host organisms must have evolved some mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Those controlling mechanisms are of general importance for understanding symbiosis (6, 10).Stinkbugs, belonging to the insect order Hemiptera, consist of over 40,000 described species in the world (15). The majority of the stinkbugs suck plant sap or tissues, and some of them are notorious as devastating agricultural pests (16). These plant-sucking stinkbugs possess a specialized symbiotic organ in their alimentary tract: A posterior region of the midgut is morphologically differentiated with a number of sacs or tubular outgrowths, called crypts or ceca, whose inner cavity hosts symbiotic bacteria (17–21). Usually, a single bacterial species dominates in the midgut crypts, and elimination of the symbiont causes retarded growth and increased mortality of the host, which indicates the specific and beneficial nature of the stinkbug gut symbiosis (20–31). The initial symbiont infection is established by nymphal feeding, which may be either via vertical transmission from symbiont-containing maternal secretion supplied upon oviposition (19–21) or via environmental acquisition from ambient microbiota (21–23). What mechanisms underlie the selective establishment of a specific bacterial symbiont in the midgut symbiotic organ despite the oral inoculum contaminated by nonsymbiotic microbes has remained largely an enigma, although recent studies have started to shed light on the symbiotic mechanisms underlying the environmental acquisition of specific Burkholderia symbionts in the bean bug Riptortus pedestris (Hemiptera: Alydidae) (22, 32). Antimicrobial substances produced by the midgut epithelia (33, 34) and some symbiont factors, such as stress-responsive polyester accumulation, cell wall synthesis, and purine biosynthesis (35–37) might be involved in the selective infection of the Burkholderia symbiont to the midgut crypts.Here we address this important symbiotic issue by the discovery of a previously unrecognized intestinal organ in the stinkbugs. Though very tiny and inconspicuous, the organ governs the configuration and specificity of the stinkbug gut symbiosis. Lying in the middle of the midgut, the organ blocks food flow and nonsymbiotic bacteria but selectively allows passing of specific symbiotic bacteria, whereby the stinkbug’s intestine is functionally partitioned into the anterior region specialized for digestion and absorption and the posterior region dedicated to symbiosis. The blocking of food flow by the organ is conserved across diverse stinkbug families, suggesting the possibility that the organ evolved in their common ancestor and has played substantial roles in their symbiont-mediated adaptation and diversification.     

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.Many dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1–6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7–9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the short-tail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From low-resolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-arm, E-loop, A-domain, and P-domain, which together place the gp10 protein in the HK97 fold category (2, 13, 14). The T7 procapsid, capsid I, contains 110–140 molecules of scaffolding protein, gp9 (4, 15, 16). After scaffolding protein expulsion the spherical T7 capsid I expands to more angular intermediates, which are collectively called capsid II (2, 4, 14, 16–18).Two DNA-free capsid IIs are purified in quantity sufficient for structural studies by cryo-EM (16). Both are produced during the normal process of wild-type T7 DNA packaging in vivo. One has an unusually low density during buoyant density centrifugation in a metrizamide density gradient (1.086 g/mL; metrizamide low density, or MLD, capsid II) and the other has a density as expected for hydrated proteins (1.28 g/mL; metrizamide high density, or MHD, capsid II) (16). The low density of MLD capsid II is caused by impermeability to metrizamide (789 Da) (16). The MLD capsid II particles are produced before MHD capsid II particles based on kinetic studies (16).The DNA packaging of T7 phage starts at capsid I state where the DNA is packaged by the ATPases (gp18 and gp19) to pass through the portal (gp8) apparatus (19). By analyzing kinetics of in vivo-produced capsids, MLD capsid II was found to be the first postcapsid I capsid. MLD capsid II appears with the kinetics of an intermediate (16) but is obviously no longer in the DNA packaging pathway because it has detached from the DNA molecule that it was packaging. MLD capsid II is not produced when a nonpermissive host is infected with a T7 amber mutant defective in DNA packaging (summarized in ref. 16). Thus, MLD capsid II is an intermediate that has been altered during either cellular lysis or subsequent purification. MHD capsid II also has the appearance kinetics of an intermediate of packaging, but one that occurs later (16). Whereas MLD capsid II has the internal core stack including proteins gp8, gp14, gp15, and gp16 (16), MHD capsid II does not have the internal core stack proteins, which were presumably lost when packaged DNA exited the capsid (16).The existence of these various capsids provides an opportunity to obtain a high-resolution (3–4 Å) analysis of structural dynamics that occur in vivo. Here we report cryo-EM structures of the shells of the following bacteriophage T7 capsids: capsid I (4.6 Å), MLD capsid II (3.5 Å), MHD capsid II (6.6 Å), and phage (3.6 Å). The two capsid II shells are the first postprocapsid, in vivo-generated shells (for any packaging system) to be subjected to high-resolution structural analysis, to our knowledge. The results reveal (i) an HK97-fold shell protein with an intracapsomere, noncovalent topological linking and another intercapsomere, joint interaction, neither interaction having been found for other dsDNA tailed phages; (ii) details of the interaction of gp9 scaffolding protein with the inner surface of the capsid I shell; (iii) a novel refolding and externalization of the N terminus of major capsid protein, gp10; and (iv) a subtle, surprising contraction of the gp10 shell in transit from MLD capsid II to phage. Based on these observations, we propose a general procapsid assembly and maturation pathway for dsDNA viruses.     

Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.     

Hierarchical self-assembly of polydisperse colloidal bananas into a two-dimensional vortex phase

Self-assembly of microscopic building blocks into highly ordered and functional structures is ubiquitous in nature and found at all length scales. Hierarchical structures formed by colloidal building blocks are typically assembled from monodisperse particles interacting via engineered directional interactions. Here, we show that polydisperse colloidal bananas self-assemble into a complex and hierarchical quasi–two-dimensional structure, called the vortex phase, only due to excluded volume interactions and polydispersity in the particle curvature. Using confocal microscopy, we uncover the remarkable formation mechanism of the vortex phase and characterize its exotic structure and dynamics at the single-particle level. These results demonstrate that hierarchical self-assembly of complex materials can be solely driven by entropy and shape polydispersity of the constituting particles.

Self-assembly of microscopic building blocks is a powerful route for preparing materials with predesigned structure and engineered properties (1–7). Nature provides a fascinating range of self-assembled architectures offering insight into how structural organization can emerge at different length scales (8–13). In the biological world, for instance, tobacco mosaic virus coat proteins self-organize into sophisticated capsids around viral RNA strands (11, 14). In molecular systems, lipid molecules, such as fatty acids, form a range of self-assembled structures as relevant as cell membranes and vesicles (15, 16). At the colloidal scale, a rich variety of crystals with remarkable optical properties, such as opal and other gemstones, also assembles from a range of colloidal constituents (12, 17–20). The structural complexity of self-assembled materials is typically dictated by the combination of the type of interactions between the constituent building blocks and their shape (2, 3, 5, 6). Colloids are ideal systems to independently study the role of these key parameters, as their shape and interactions can be systematically tuned and rationally designed (5, 18, 21–23).In colloidal systems interacting solely via excluded volume interactions, the shape of the particles can already lead to the assembly of complex structures (24–28). For instance, binary colloidal crystals (25) are obtained from spherical particles, complex dodecagonal quasicrystals are formed by tetrahedrons (26), and exotic banana-shaped liquid crystals are assembled from colloidal bananas (28). Introducing complex interactions between the colloidal building blocks—on the top of their shape—leads to their assembly into hierarchical materials with structural order at multiple length scales (3, 29–31). Examples include colloidal diamond structures assembled by patchy tetrahedrons functionalized with DNA strands (20) and superlattice structures formed by octapod-like particles functionalized with hydrophobic molecules (32). The successful hierarchical self-assembly of these structures relies not only on the directionality of the particle interactions but also, on the uniformity in size of the constituent building blocks, as polydispersity typically disrupts ordering via the formation of defects (33, 34).In this work, however, we show that a colloidal suspension of polydisperse banana-shaped particles interacting only via simple excluded volume interactions (28) self-assembles into remarkably ordered concentric structures, which we term colloidal vortices. At high packing fractions, these structures form a quasi–two-dimensional (quasi-2D) hierarchical material, which we term the vortex phase. Using confocal microscopy, we uncover the formation mechanism of this tightly packed phase and characterize its exotic structure and dynamics at the single-particle level.     

The NMR–Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope

Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6–16.7 Å and a tilt of 36.1–36.6° between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magic-angle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.Filamentous bacteriophage are long, thin, and semiflexible rod viruses that infect bacteria (1, 2). These large assemblies (∼15–35 MDa) contain a circular single-stranded (ss) DNA genome encapsulated in a protein shell. All filamentous phage have a similar life cycle and virion structure despite the relatively high number of strains, with DNA sequence homology varying from almost complete to very little. The unique phage properties make them ideal for a large range of applications such as phage display (3), DNA cloning and sequencing (4, 5), nanomaterial fabrication (6–8), and as drug-carrying nanomachines (9). In addition, filamentous viruses form a variety of liquid crystals driving the development of both theory and practice of soft-matter physics (10, 11). Filamentous viruses are also associated with various diseases, e.g., CTXϕ phage in cholera toxin (12) and Pf4 phage in cystic fibrosis (13).Phage belonging to the Ff family (M13, fd, f1) are F-pilus–specific viruses that share almost identical genomes and very similar structures. M13 is a 16-MDa virus having a diameter of ∼7 nm and a length of ∼1 μm. The capsid is composed of several thousand identical copies of a major coat protein subunit arranged in a helical array surrounding a core of a circular ssDNA. The major coat proteins constitute ∼85% of the total virion mass, the ssDNA ∼12%, and all other minor proteins (gp3, gp6, gp7, gp9) that are specific for infection and assembly constitute about 3% of the total virion mass (1, 14).Previous structural models for a small number of phages have been obtained by means of X-ray fiber diffraction (15–19), static solid-state NMR (20, 21), and cryo-EM (22). Structural models for the Ff family have been proposed based on the three methods; however, satisfactory resolution was only obtained for the Y21M mutant of the fd phage (17, 18, 21) (wt fd is related to M13 by one additional mutation, N12D). The only reported model for M13 (23) (no coordinates available) and models of fd-Y21M from different methods differ in detail (24) and lack accuracy in some structural details such as the N-terminus orientation, the nature of DNA–protein interactions, and sidechain interactions, which are the dominant packing elements of the capsid. The most recent model was built using a combination of static NMR and fiber diffraction (17).According to fiber diffraction, the symmetry of the Ff capsid is C₅S₂, also referred to as class I symmetry. That is, a fivefold rotation of the major coat protein subunit around the virion axis (pentamers) and an approximate 36° rotation relating two successive pentamers [in fd-Y21M a precise 36° rotation was reported; for fd, values of −33.23° (18) and −34.62° (22) were reported]. All studies report that the coat protein is mostly right-handed, curved, α-helical, with a flexible or disordered N terminus.Magic-angle spinning (MAS) solid-state NMR has become a popular tool for studying the structure and dynamics of biological molecules (25–27). The method can be implemented on a variety of systems from small peptides to macromolecular biological assemblies. Integrated approaches can be used to resolve structures of large assemblies (28, 29) and recently, the combination of MAS NMR data, cryo-EM, and Rosetta modeling resulted in a detailed atomic structure of the recombinant type III secretion system needle (30, 31). We have previously performed MAS NMR studies on both wt fd and M13 in a precipitated form (32–34). Their chemical shifts pointed to a single homogeneous capsid subunit that is mostly helical and curved with a mobile N terminus. NMR studies of the interactions between the capsid and the DNA reported on the subunit orientation with respect to the viral axis, and indicated that the C terminus undergoes electrostatic interactions with the DNA.In this study, we use homonuclear 2D ¹³C–¹³C correlation experiments on sparsely labeled M13 samples together with our prior backbone and sidechain resonance assignments of the M13 phage to acquire MAS NMR structure restraints. Using the CS-Rosetta fold-and-dock protocol (35) we derive an atomic detailed well-converged quaternary structural model of the intact M13 phage viral capsid. The specific bacteriophage symmetry produces four identical, repeating hydrophobic pockets for each subunit, resulting in tight subunit packing that stabilizes the phage assembly.