A stapled lipopeptide platform for preventing and treating highly pathogenic viruses of pandemic potential.

The continued emergence of highly pathogenic viruses, which either thwart immune- and small molecule-based therapies or lack interventions entirely, mandates alternative approaches, particularly for prompt and facile pre- and post-exposure prophylaxis. Many highly pathogenic viruses, including coronaviruses, employ the six-helix bundle heptad repeat membrane fusion mechanism to achieve infection. Although heptad-repeat-2 decoys can inhibit viral entry by blocking six-helix bundle assembly, the biophysical and pharmacologic liabilities of peptides have hindered their clinical development. Here, we develop a chemically stapled lipopeptide inhibitor of SARS-CoV-2 as proof-of-concept for the platform. We show that our lead compound blocks infection by a spectrum of SARS-CoV-2 variants, exhibits mucosal persistence upon nasal administration, demonstrates enhanced stability compared to prior analogs, and mitigates infection in hamsters. We further demonstrate that our stapled lipopeptide platform yields nanomolar inhibitors of respiratory syncytial, Ebola, and Nipah viruses by targeting heptad-repeat-1 domains, which exhibit strikingly low mutation rates, enabling on-demand therapeutic intervention to combat viral outbreaks.

Role of DNA-DNA sliding friction and nonequilibrium dynamics in viral genome ejection and packaging.

Many viruses eject their DNA via a nanochannel in the viral shell, driven by internal forces arising from the high-density genome packing. The speed of DNA exit is controlled by friction forces that limit the molecular mobility, but the nature of this friction is unknown. We introduce a method to probe the mobility of the tightly confined DNA by measuring DNA exit from phage phi29 capsids with optical tweezers. We measure extremely low initial exit velocity, a regime of exponentially increasing velocity, stochastic pausing that dominates the kinetics and large dynamic heterogeneity. Measurements with variable applied force provide evidence that the initial velocity is controlled by DNA-DNA sliding friction, consistent with a Frenkel-Kontorova model for nanoscale friction. We confirm several aspects of the ejection dynamics predicted by theoretical models. Features of the pausing suggest that it is connected to the phenomenon of 'clogging' in soft matter systems. Our results provide evidence that DNA-DNA friction and clogging control the DNA exit dynamics, but that this friction does not significantly affect DNA packaging.

Particle generation and dispersion from high-speed dental drilling.

OBJECTIVE: To investigate the characteristics of particle generation and dispersion during dental procedure using digital inline holography (DIH) METHODS: Particles at two locations, near-field and far-field, which represent the field closer to the procedure location and within 0.5 m from the procedure location respectively, are studied using two different DIH systems. The effect of three parameters namely rotational speed, coolant flow rate, and bur angle on particle generation and dispersion are evaluated by using 10 different operating conditions. The particle characteristics at different operating conditions are estimated from the holograms using machine learning-based analysis. RESULTS: The particle concentration decreased by at least two orders of magnitude between the near-field and far-field locations across the 10 different operating conditions, indicating significant dispersion of the particles. High rotational speed is found to produce a larger number of smaller particles, while lower rotational speeds generate larger particles. Coolant flow rate is found to have a greater impact on particle transport to the far-field location. Irregular shape dental particles account for 29% of total particles at far-field location, with the majority of these irregular shape particles having diameters ranging from 12 to 18 µm. CONCLUSIONS: All three parameters have significant effects on particle generation and dispersion, with rotational speed having a more significant influence on particle generation at near-field and coolant flow rate playing a more important role on particle transport to the far-field. CLINICAL RELEVANCE: This study provides valuable insights on particle characteristics during high-speed drilling. It can help dental professionals minimize exposure risks for themselves and patients by optimizing clinical operating conditions.

Role of DNA-DNA sliding friction and non-equilibrium dynamics in viral genome ejection and packaging.

Many viruses eject their DNA via a nanochannel in the viral shell, driven by internal forces arising from the high-density genome packing. The speed of DNA exit is controlled by friction forces that limit the molecular mobility, but the nature of this friction is unknown. We introduce a method to probe the mobility of the tightly confined DNA by measuring DNA exit from phage phi29 capsids with optical tweezers. We measure extremely low initial exit velocity, a regime of exponentially increasing velocity, stochastic pausing that dominates the kinetics, and large dynamic heterogeneity. Measurements with variable applied force provide evidence that the initial velocity is controlled by DNA-DNA sliding friction, consistent with a Frenkel-Kontorova model for nanoscale friction. We confirm several aspects of the ejection dynamics predicted by theoretical models. Features of the pausing suggest it is connected to the phenomenon of "clogging" in soft-matter systems. Our results provide evidence that DNA-DNA friction and clogging control the DNA exit dynamics, but that this friction does not significantly affect DNA packaging.

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

Kinetics of ATP/ADP binding to the gp16 ATPase.

The gp16 ATPase is the constituent subunit of the pentameric dsDNA (double-stranded deoxyribonucleic acid) translocation motor of the Bacillus subtilis Φ29 bacteriophage. Although recent single-molecule studies have provided tantalizing clues about the activity of this motor, the mechanism by which the gp16 subunits couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of dsDNA translocation remains unknown. To address this need, we have characterized the binding of fluorophore-labeled ATP and ADP to monomeric gp16 using a stopped-flow fluorescence assay. These experiments show that the binding of ATP/ADP occurs through a single-step mechanism with corresponding affinities of 523.8 ± 247.3 nM for ATP and a lower limit of 30 µM for ADP. When analyzed through the lens of changes in free energy of the system, this difference in binding affinities is reasonable for a cyclical process of binding, hydrolysis, and product release. In addition to answering questions about the activity of monomeric gp16, these results are also a necessary step in constructing a model for intersubunit communication within the pentameric gp16 motor.

TTC39B destabilizes retinoblastoma protein promoting hepatic lipogenesis in a sex-specific fashion.

BACKGROUND & AIMS: Molecular mechanisms underlying the different susceptibility of men and women to non-alcoholic fatty liver disease (NAFLD) are poorly understood. The TTC39B locus encodes a scaffolding protein, associates with gynecological disorders and its deletion protects mice from diet-induced steatohepatitis. This study aimed to elucidate the molecular mechanisms linking TTC39B (T39) to the expression of lipogenic genes and to explore sex-specific effects. METHODS: Co-expression in HEK293A cells validated the novel T39/pRb interaction predicted by a protein-protein interaction algorithm. T39 was knocked down using an antisense oligonucleotide (ASO) in mice with dietary NAFLD and a genetic deficiency of pRb or its downstream effector E2F1, as well as in primary human hepatocytes. RESULTS: T39 interacts with pRb via its C-terminal TPR domain and promotes its proteasomal degradation. In female mice, T39 deficiency reduces the mRNA of lipogenic genes, especially Pnpla3, in a pRb- and E2F1-dependent manner. In contrast, in male mice, T39 deficiency results in a much smaller reduction in lipogenic gene expression that is independent of pRb/E2F1. T39 also interacts with VAPB via an N-terminal FFAT motif and stabilizes the interaction of VAPB with SCAP. Ovariectomy abolishes the effect of T39 knockdown on the hepatic pRb/E2F1/Pnpla3 axis. In both sexes T39 knockdown reduces SCAP independently of pRb. In primary human hepatocytes, T39 knockdown reduces expression of PNPLA3 and other lipogenic genes in women but not men. CONCLUSIONS: We have uncovered a conserved sexual dimorphism in the regulation of hepatic lipogenic genes, with effects of T39 mediated through pRb/E2F1 in females and VAPB/SCAP in both sexes. T39 inhibition could be a novel strategy to downregulate PNPLA3 and treat NAFLD in women. LAY SUMMARY: In females, the protein TTC39B degrades a tumor suppressor in the liver to promote the synthesis of new fat and the expression of a major genetic risk factor for non-alcoholic fatty liver disease. TTC39B is a potential therapeutic target for non-alcoholic fatty liver disease, especially in women.

Characterization and mitigation of aerosols and spatters from ultrasonic scalers.

BACKGROUND: Dental procedures often produce aerosols and spatter, which have the potential to transmit pathogens such as severe acute respiratory syndrome coronavirus 2. The existing literature is limited. METHODS: Aerosols and spatter were generated from an ultrasonic scaling procedure on a dental manikin and characterized via 2 optical imaging methods: digital inline holography and laser sheet imaging. Capture efficiencies of various aerosol mitigation devices were evaluated and compared. RESULTS: The ultrasonic scaling procedure generated a wide size range of aerosols (up to a few hundred µm) and occasional large spatter, which emit at low velocity (mostly < 3 m/s). Use of a saliva ejector and high-volume evacuator (HVE) resulted in overall reductions of 63% and 88%, respectively, whereas an extraoral local extractor (ELE) resulted in a reduction of 96% at the nominal design flow setting. CONCLUSIONS: The study results showed that the use of ELE or HVE significantly reduced aerosol and spatter emission. The use of HVE generally requires an additional person to assist a dental hygienist, whereas an ELE can be operated hands free when a dental hygienist is performing ultrasonic scaling and other operations. PRACTICAL IMPLICATIONS: An ELE aids in the reduction of aerosols and spatters during ultrasonic scaling procedures, potentially reducing transmission of oral or respiratory pathogens like severe acute respiratory syndrome coronavirus 2. Position and airflow of the device are important to effective aerosol mitigation.

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures.

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA.

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM-focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor.

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.

Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation.

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.

NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors.

Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

ATP/ADP modulates gp16-pRNA conformational change in the Phi29 DNA packaging motor.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA-gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a 'Z'-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

Slow and steady wins the race: physical limits on the rate of viral DNA packaging.

During the assembly of dsDNA viruses such as the tailed bacteriophages and herpesviruses, the viral chromosome is compacted to near crystalline density inside a preformed head shell. DNA translocation is driven by powerful ring ATPase motors that couple ATP binding, hydrolysis, and release to force generation and movement. Studies of the motor of the bacteriophage phi29 have revealed a complex mechanochemistry behind this process that slows as the head fills. Recent studies of the physical behavior of packaging DNA suggest that surprisingly long-time scales of relaxation of DNA inside the head and jamming phenomena during packaging create the physical need for regulation of the rate of packaging. Studies of DNA packaging in viral systems have, therefore, revealed fundamental insight into the complex behavior of DNA and the need for biological systems to accommodate these physical constraints.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

RATIONALE: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction.

The bottom-up construction of biological entities from genetic information provides a broad range of opportunities to better understand fundamental processes within living cells, as well as holding great promise for the development of novel biomedical applications. Cell-free transcription-translation (TXTL) systems have become suitable platforms to tackle such topics because they recapitulate the process of gene expression. TXTL systems have advanced to where the in vitro construction of viable, complex, self-assembling deoxyribonucleic acid-programmed biological entities is now possible. Previously, we demonstrated the cell-free synthesis of three bacteriophages from their genomes: MS2, ΦX174, T7. In this work, we present the complete synthesis of the phage T4 from its 169-kbp genome in one-pot TXTL reactions. This achievement, for one of the largest coliphages, demonstrates the integration of complex gene regulation, metabolism and self-assembly, and brings the bottom-up synthesis of biological systems to a new level.