Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.

Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2.

Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.

Molecular Basis of Arthritogenic Alphavirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein.

Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

The mechanism of STING autoinhibition and activation.

2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.

Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

Structural insights into the molecular effects of the anthelmintics monepantel and betaine on the Caenorhabditis elegans acetylcholine receptor ACR-23.

Anthelmintics are drugs used for controlling pathogenic helminths in animals and plants. The natural compound betaine and the recently developed synthetic compound monepantel are both anthelmintics that target the acetylcholine receptor ACR-23 and its homologs in nematodes. Here, we present cryo-electron microscopy structures of ACR-23 in apo, betaine-bound, and betaine- and monepantel-bound states. We show that ACR-23 forms a homo-pentameric channel, similar to some other pentameric ligand-gated ion channels (pLGICs). While betaine molecules are bound to the classical neurotransmitter sites in the inter-subunit interfaces in the extracellular domain, monepantel molecules are bound to allosteric sites formed in the inter-subunit interfaces in the transmembrane domain of the receptor. Although the pore remains closed in betaine-bound state, monepantel binding results in an open channel by wedging into the cleft between the transmembrane domains of two neighboring subunits, which causes dilation of the ion conduction pore. By combining structural analyses with site-directed mutagenesis, electrophysiology and in vivo locomotion assays, we provide insights into the mechanism of action of the anthelmintics monepantel and betaine.

Universal inverse modeling of point spread functions for SMLM localization and microscope characterization.

The point spread function (PSF) of a microscope describes the image of a point emitter. Knowing the accurate PSF model is essential for various imaging tasks, including single-molecule localization, aberration correction and deconvolution. Here we present universal inverse modeling of point spread functions (uiPSF), a toolbox to infer accurate PSF models from microscopy data, using either image stacks of fluorescent beads or directly images of blinking fluorophores, the raw data in single-molecule localization microscopy (SMLM). Our modular framework is applicable to a variety of microscope modalities and the PSF model incorporates system- or sample-specific characteristics, for example, the bead size, field- and depth- dependent aberrations, and transformations among channels. We demonstrate its application in single or multiple channels or large field-of-view SMLM systems, 4Pi-SMLM, and lattice light-sheet microscopes using either bead data or single-molecule blinking data.

Structural insight into arenavirus replication machinery.

Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.

Cryo-EM structure of DNA polymerase of African swine fever virus.

African swine fever virus (ASFV) is one of the most important causative agents of animal diseases and can cause highly fatal diseases in swine. ASFV DNA polymerase (DNAPol) is responsible for genome replication and highly conserved in all viral genotypes showing an ideal target for drug development. Here, we systematically determined the structures of ASFV DNAPol in apo, replicating and editing states. Structural analysis revealed that ASFV DNAPol had a classical right-handed structure and showed the highest similarity to the structure of human polymerase delta. Intriguingly, ASFV DNAPol has a much longer fingers subdomain, and the thumb and palm subdomain form a unique interaction that has never been seen. Mutagenesis work revealed that the loss of this unique interaction decreased the enzymatic activity. We also found that the ß-hairpin of ASFV DNAPol is located below the template strand in the editing state, which is different from the editing structures of other known B family DNAPols with the ß-hairpin above the template strand. It suggests that B family DNAPols have evolved two ways to facilitate the dsDNA unwinding during the transition from replicating into editing state. These findings figured out the working mechanism of ASFV DNAPol and will provide a critical structural basis for the development of antiviral drugs.

Structural basis for difunctional mechanism of m-AMSA against African swine fever virus pP1192R.

The African swine fever virus (ASFV) type II topoisomerase (Topo II), pP1192R, is the only known Topo II expressed by mammalian viruses and is essential for ASFV replication in the host cytoplasm. Herein, we report the structures of pP1192R in various enzymatic stages using both X-ray crystallography and single-particle cryo-electron microscopy. Our data structurally define the pP1192R-modulated DNA topology changes. By presenting the A2+-like metal ion at the pre-cleavage site, the pP1192R-DNA-m-AMSA complex structure provides support for the classical two-metal mechanism in Topo II-mediated DNA cleavage and a better explanation for nucleophile formation. The unique inhibitor selectivity of pP1192R and the difunctional mechanism of pP1192R inhibition by m-AMSA highlight the specificity of viral Topo II in the poison binding site. Altogether, this study provides the information applicable to the development of a pP1192R-targeting anti-ASFV strategy.

Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.

CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.

Modeling global oceanic nitrogen deposition from food systems and its mitigation potential by reducing overuse of fertilizers.

Growing population and consumption pose unprecedented demands on food production. However, ammonia emissions mainly from food systems increase oceanic nitrogen deposition contributing to eutrophication. Here, we developed a long-term oceanic nitrogen deposition dataset (1970 to 2018) with updated ammonia emissions from food systems, evaluated the impact of ammonia emissions on oceanic nitrogen deposition patterns, and discussed the potential impact of nitrogen fertilizer overuse. Based on the chemical transport modeling approach, oceanic ammonia-related nitrogen deposition increased by 89% globally between 1970 and 2018, and now, it exceeds oxidized nitrogen deposition by over 20% in coastal regions including China Sea, India Coastal, and Northeastern Atlantic Shelves. Approximately 38% of agricultural nitrogen fertilizer was excessive, which corresponds to 15% of global oceanic ammonia-related nitrogen deposition. Policymakers and water quality managers need to pay increasingly more attention to ammonia associated with food production if the goal of reducing coastal nitrogen pollution is to be achieved for Sustainable Development Goals.

Broad protective RBD heterotrimer vaccines neutralize SARS-CoV-2 including Omicron sub-variants XBB/BQ.1.1/BF.7.

SARS-CoV-2 variants with severe immune evasion are a major challenge for COVID-19 prevention, especially the circulating Omicron XBB/BQ.1.1/BF.7 strains. Thus, the next-generation of broad-spectrum vaccines are urgently needed. Previously, we developed a COVID-19 protein subunit vaccine, ZF2001, based on the RBD-homodimer as the immunogen. To adapt SARS-CoV-2 variants, we developed chimeric RBD-heterodimers to induce broad immune responses. In this study, we further explored the concept of tandem RBD homotrimer and heterotrimer. Prototype SARS-CoV-2 RBD-homotrimer, prototype-Delta-BA.1 (PDO) RBD-heterotrimer and Delta-BA.2-BA.5 (DBA2BA5) RBD-heterotrimer were designed. Biochemical and cryo-EM structural characterization demonstrated total epitope exposure of the RBD-trimers. In mouse experiments, PDO and DBA2BA5 elicited broad SARS-CoV-2 neutralization. Potent protection against SARS-CoV-2 variants was observed in challenge assays and was correlated with neutralizing antibody titer. This study validated the design strategy of tandem RBD-heterotrimers as multivalent immunogens and presented a promising vaccine candidate, DBA2BA5, eliciting broad-spectrum immune responses, including against the circulating XBB/BF.7/BQ.1.1.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

Cylindrical Vector Beam Holography without Preservation of OAM Modes.

Orbital angular momentum (OAM) multiplexed holograms have attracted a great deal of attention recently due to their physically unbounded set of orthogonal helical modes. However, preserving the OAM property in each pixel hinders fine sampling of the target image in principle and requires a fundamental filtering aperture array in the detector plane. Here, we demonstrate the concept of metasurface-based vectorial holography with cylindrical vector beams (CVBs), whose unlimited polarization orders and unique polarization distributions can be used to boost information storage capacity. Although CVBs are composed of OAM modes, the holographic images do not preserve the OAM modes in our design, enabling fine sampling of the target image in a quasi-continuous way like traditional computer-generated holograms. Moreover, the images can be directly observed by passing them through a polarizer without the need for a fundamental mode filter array. We anticipate that our method may pave the way for high-capacity holographic devices.

Synthesis of Ultrathin High-Entropy Oxides with Phase Controllability.

High-entropy oxides (HEOs) with an ultrathin geometric structure are especially expected to exhibit extraordinary performance in different fields. The phase structure is deemed as a key factor in determining the properties of HEOs, rendering their phase control synthesis tempting. However, the disparity in intrinsic phase structures and physicochemical properties of multiple components makes it challenging to form single-phase HEOs with the target phase. Herein, we proposed a self-lattice framework-guided strategy to realize the synthesis of ultrathin HEOs with desired phase structures, including rock-salt, spinel, perovskite, and fluorite phases. The participation of the Ga assistor was conducive to the formation of the high-entropy mixing state by decreasing the formation energy. The as-prepared ultrathin spinel HEOs were demonstrated to be an excellent catalyst with high activity and stability for the oxygen evolution reaction in water electrolysis. Our work injects new vitality into the synthesis of HEOs for advanced applications and undoubtedly expedites their phase engineering.

LncRNA HBL1 is required for genome-wide PRC2 occupancy and function in cardiogenesis from human pluripotent stem cells.

Polycomb repressive complex 2 (PRC2) deposits H3K27me3 on chromatin to silence transcription. PRC2 broadly interacts with RNAs. Currently, the role of the RNA-PRC2 interaction in human cardiogenesis remains elusive. Here, we found that human-specific heart brake lncRNA 1 (HBL1) interacted with two PRC2 subunits, JARID2 and EED, in human pluripotent stem cells (hPSCs). Loss of JARID2, EED or HBL1 significantly enhanced cardiac differentiation from hPSCs. HBL1 depletion disrupted genome-wide PRC2 occupancy and H3K27me3 chromatin modification on essential cardiogenic genes, and broadly enhanced cardiogenic gene transcription in undifferentiated hPSCs and later-on differentiation. In addition, ChIP-seq revealed reduced EED occupancy on 62 overlapped cardiogenic genes in HBL1-/- and JARID2-/- hPSCs, indicating that the epigenetic state of cardiogenic genes was determined by HBL1 and JARID2 at pluripotency stage. Furthermore, after cardiac development occurs, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved 'microRNA-1-JARID2' axis to modulate cardiogenic gene transcription. Overall, our findings delineate the indispensable role of HBL1 in guiding PRC2 function during early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to orchestrate human cardiogenesis.

Self-Cascade Ce-MOF-818 Nanozyme for Sequential Hydrolysis and Oxidation.

Mimicking efficient biocatalytic cascades using nanozymes has gained enormous attention in catalytic chemistry, but it remains challenging to develop a nanozyme-based cascade system to sequentially perform the desired reactions. Particularly, the integration of sequential hydrolysis and oxidation reactions into nanozyme-based cascade systems has not yet been achieved, despite their significant roles in various domains. Herein, a self-cascade Ce-MOF-818 nanozyme for sequential hydrolysis and oxidation reactions is developed. Ce-MOF-818 is the first Ce(IV)-based heterometallic metal-organic framework constructed through the coordination of Ce and Cu to distinct groups. It is successfully synthesized using an improved solvothermal method, overcoming the challenge posed by the significant difference in the binding speeds of Ce and Cu to ligands. With excellent organophosphate hydrolase-like (Km = 42.3 µM, Kcat = 0.0208 min-1 ) and catechol oxidase-like (Km = 2589 µM, Kcat = 1.25 s-1 ) activities attributed to its bimetallic active centers, Ce-MOF-818 serves as a promising self-cascade platform for sequential hydrolysis and oxidation. Notably, its catalytic efficiency surpasses that of physically mixed nanozymes by approximately fourfold, owning to the close integration of active sites. The developed hydrolysis-oxidation self-cascade nanozyme has promising potential applications in catalytic chemistry and provides valuable insights into the rational design of nanozyme-based cascade systems.