Cholesterol (CHL) serves as a building block for membrane biogenesis and a precursor to oxysterols, steroid hormones, bile acids, and vitamin D. The lysosome serves as a major sorting station for low-density lipoproteins (LDLs), which carry dietary CHL, and it is also the cellular site where the master growth regulator, the protein kinase mechanistic Target of Rapamycin Complex 1 (mTORC1), is activated. Recently, the lysosomal transmembrane protein GPR155 was reported to signals CHL sufficiency to mTORC1 through sequestration of the GTPase-activating protein towards the Rags 1 (GATOR1). Although the recently reported structures of GPR155 have revealed the CHL binding site, how the signal is transduced from the CHL binding site to the soluble parts of GPR155 and GATOR1 remains unknown. Here, with our three cryo-EM structures of GPR155 captured in different conformations in complex with CHL, complemented by long-time scale molecular dynamics simulations, the dynamic rearrangement of different domains was observed. CHL binding induces a widening of the crevice between the transporter and GPCR domains. The extending helix preceding transmembrane helix (TM) 16, which was unresolved in other structures, acts as a linkage lever that transmits the rotation of the GPCR domain to the soluble parts of GPR155 in response to CHL binding. This work not only answers the question of how CHL is sensed by GPR155, but also addresses a more profound question: how the signal perceived by the TMs regions is transduced to the LED and DEP domains.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously evolving since 2019. Some monoclonal antibodies (mAbs) have been developed and widely used, such as etesevimab (CB6) developed by Eli-Lilly/Junshi. However, the mAb escaped from the variant of concern (VOC) ever since the emergence of Beta VOC, with a complete loss of efficacy against the Omicron subvariants. Here, we developed a broad-spectrum and affinity-mature antibody design (BAADesign) procedure to design CB6, enabling it to bind to the receptor-binding domains (RBDs) of multiple important Omicron subvariants, including the recent variant KP.2. Structural analysis confirmed the desired CB6-RBD interactions. Additionally, identical mutations in the complementarity determining regions (CDR)1 and CDR2 of the CB6 mutants also restored neutralizing potency for some RBD-1 group antibodies. Overall, the enhanced CB6 neutralizing capacity makes it a promising candidate against SARS-CoV-2 infection, and the BAADesign method has implications for the design of other antibodies.

Plasma C-reactive protein (CRP) is widely used as a biomarker for bacterial infections due to its massive induction during infections. However, the biological function of CRP remains largely undefined. Here we show that CRP enables liver resident macrophages (Kupffer cells) to capture and eliminate a wide range of invasive bacteria from the bloodstream of mice, and thereby provides rapid and sterilizing immunity. Mechanistically, CRP binds to at least 20 capsule types of Gram-positive and -negative pathogens, and shuffles the encapsulated bacteria to Kupffer cells embedded in the lining of the liver sinusoidal vasculatures by the complement-dependent and -independent pathways. The complement-dependent mode involves the activation of complement C3 at the bacterial surface, and the capture of the C3-opsonized bacteria by the CRIg and CR3 complement receptors on Kupffer cells. Cryo-electron microscopy analysis revealed a flexible structural framework for CRP’s recognition of structurally diverse capsular polysaccharides. Because human CRP also possesses the broad capsule-binding activities, our findings provide a biological reason for the massive rise of plasma CRP during bacterial infections.

In 2018, two novel influenza-like virus genomes were first identified in basal vertebrates: the Asiatic toads (Bufo gargarizans) and spiny eels (Mastacembelus aculeatus). Their hemagglutinin (HA) proteins exhibit remarkably low amino acid sequences homology (23.0% and 42.8%, respectively) compared to influenza B virus (IBV), their closest canonical influenza virus relative. This study revealed that the Asiatic toad influenza-like virus HA (tHA) demonstrates dual receptor specificity, bound both α2-3 (avian-type) and α2-6 (human-type) sialic acid (SA) receptors, whereas the spiny eel influenza-like virus HA (eHA) lacks this capability. Biophysical characterization showed reduced thermal stability (lower Tm values) for both tHA and eHA compared to canonical influenza HA. Furthermore, we determined the cryo-EM structures of apo-tHA, tHA in complex with either α2-3 SA receptor or α2-6 SA receptor, as well as apo-eHA and eHA bound to GM2 complex. Our analysis revealed that tHA has a shorter length and looser HA trimer packing compared to canonical HA. These findings collectively indicate that influenza-like viruses in basal vertebrates have evolutionarily acquired dual SA receptor-binding capacity, a trait critical for cross-species transmission in influenza viruses. However, the observed thermolability of these HA proteins suggests that host physiological temperatures may impose a barrier to zoonotic spillover.

The Mpox virus (MPXV) is an orthopoxvirus that caused a global outbreak in 2022. The poxvirus DNA polymerase complex is responsible for the replication and integrity of the viral genome; however, the molecular mechanisms underlying DNA replication fidelity are still unclear. In this study, we determined the cryoelectron microscopy (cryo-EM) structures of the MPXV F8-A22-E4 polymerase holoenzyme in its editing state, in complex with mismatched primer-template DNA and DNA containing uracil deoxynucleotide. We showed that the MPXV polymerase has a similar replication-to-edit transition mechanism to proofread the mismatched nucleotides like the B-family DNA polymerases of other species. The unique processivity cofactor A22-E4 undergoes conformational changes in different working states and might affect the proofreading process. Moreover, we elucidated the base excision repair (BER) function of E4 as a uracil-DNA glycosylase and the coupling mechanism of genome replication and BER, characteristic of poxviruses. Our findings greatly enhance our molecular understanding of DNA replication fidelity of orthopoxviruses and will stimulate the development of broad-spectrum antiviral drugs.

Recent avian-origin H3N8 influenza A virus (IAV) that have infected humans pose a potential public health concern. Alterations in the viral surface glycoprotein, hemagglutinin (HA), are typically required for IAVs to cross the species barrier for adaptation to a new host, but whether H3N8 has adapted to infect humans remains elusive. The observation of a degenerative codon in position 228 of HA in human H3N8 A/Henan/4-10/2022 protein sequence, which could be residue G or S, suggests a dynamic viral adaptation for human infection. Previously, we found this human-isolated virus has shown the ability to transmit between ferrets via respiratory droplets, with the HA-G228S substitution mutation emerging as a critical determinant for the airborne transmission of the virus in ferrets. Here, we investigated the receptor-binding properties of these two H3N8 HAs. Our results showed H3N8 HAs have dual receptor-binding properties with a preference for avian receptor binding, and G228S slightly increased binding to human receptors. Cryo-electron microscopy structures of the two H3N8 HAs with avian and human receptor analogs revealed the basis for dual receptor binding. Mutagenesis studies reveal that the Q226L mutation shifts H3N8 HA's receptor preference from avian to human, while the G228S substitution enhances binding to both receptor types. H3N8 exhibits distinct antigenic sites compared to H3N2, prompting concerns regarding vaccine efficacy. These findings suggest that the current H3N8 human isolates are yet to adapt for efficient human-to-human transmission and further continuous surveillance should be implemented.IMPORTANCEInfluenza virus transmission remains a public health concern currently. H3N8 subtype influenza A viruses infect humans and their HAs acquire the ability to bind to both human and avian receptors, posing a threat to human health. We have solved and analyzed the structural basis of dual receptor binding of recently human-infecting H3N8 HA, and we demonstrate that the G228S enhances human receptor binding and adaptation. We also found that HN/4-10 H3N8 HA has distinct antigenic sites, which challenges vaccine efficacy. Taken together, our work is critical to the prevention and control of human H3 influenza virus infection.

AbstractGenome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

Nanoparticle vaccines displaying mosaic receptor -binding domains (RBDs) or spike (S) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other sarbecoviruses are used in preparedness against potential zoonotic outbreaks. Here, we describe a self -assembling nanoparticle using lumazine synthase (LuS) as the scaffold to display RBDs from different sarbecoviruses. Mosaic nanoparticles induce sarbecovirus cross -neutralizing antibodies comparable to a nanoparticle cocktail. We find mosaic nanoparticles elicit a B cell receptor repertoire using an immunodominant germline gene pair of IGHV143:IGKV14-111 . Most of the tested IGHV14-3:IGKV14-111 monoclonal antibodies (mAbs) are broadly cross -reactive to clade 1a, 1b, and 3 sarbecoviruses. Using mAb competition and cryo-electron microscopy, we determine that a representative IGHV14-3:IGKV14-111 mAb, M2-7, binds to a conserved epitope on the RBD, largely overlapping with the pan-sarbecovirus mAb S2H97. This suggests mosaic nanoparticles expand B cell recognition of the common epitopes shared by different clades of sarbecoviruses. These results provide immunological insights into the cross -reactive responses elicited by mosaic nanoparticles against sarbecoviruses.

Plasma C-reactive protein (CRP) is widely used as a biomarker for bacterial infections due to its massive induction during infections, however, the precise function of CRP in bacterial infections remains undefined. Here we show that CRP enables Kupffer cells (liver macrophages) to capture and eliminate a wide range of encapsulated bacteria from the bloodstream of mice and thereby provides rapid and effective immunity. Mechanistically, CRP binds to the structurally diverse capsular polysaccharides of major Gram-positive and -negative pathogens, and thereby activates complement C3 at the bacterial surface. The C3-opsonized microbes are in turn captured by C3 receptors on the surface of Kupffer cells, and eliminated in the liver sinusoids. Since CRP principally shares the functional features of antibodies in pathogen recognition/execution, CRP-based defense combines the broad spectrum of the innate immunity with the swiftness, potency and specificity of the adaptive immunity, which helps explain massive rise of CRP during systemic bacterial infections. ### Competing Interest Statement The authors have declared no competing interest.

Since SARS-CoV-2 Omicron variant emerged, it is constantly evolving into multiple sub-variants, including BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5 and the recently emerged BA.2.86 and JN.1. Receptor binding and immune evasion are recognized as two major drivers for evolution of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein. However, the underlying mechanism of interplay between two factors remains incompletely understood. Herein, we determined the structures of human ACE2 complexed with BF.7, BQ.1, BQ.1.1, XBB and XBB.1.5 RBDs. Based on the ACE2/RBD structures of these sub-variants and a comparison with the known complex structures, we found that R346T substitution in the RBD enhanced ACE2 binding upon an interaction with the residue R493, but not Q493, via a mechanism involving long-range conformation changes. Furthermore, we found that R493Q and F486V exert a balanced impact, through which immune evasion capability was somewhat compromised to achieve an optimal receptor binding. We propose a "two-steps-forward and one-step-backward" model to describe such a compromise between receptor binding affinity and immune evasion during RBD evolution of Omicron sub-variants.

The 1957 H2N2 influenza pandemic virus [A(H2N2)pdm1957] has disappeared from humans since 1968, while H2N2 avian influenza viruses (AIVs) are still circulating in birds. It is necessary to reveal the recurrence risk and potential cross-species infection of these AIVs from avian to mammals. We find that H2 AIVs circulating in domestic poultry in China have genetic and antigenic differences compared to the A(H2N2)pdm1957. One H2N2 AIV has a dual receptor-binding property similar to that of the A(H2N2)pdm1957. Molecular and structural studies reveal that the N144S, and N144E or R137M substitutions in hemagglutinin (HA) enable H2N2 avian or human viruses to bind or preferentially bind human-type receptor. The H2N2 AIV rapidly adapts to mice (female) and acquires mammalian-adapted mutations that facilitated transmission in guinea pigs and ferrets (female). These findings on the receptor-binding, infectivity, transmission, and mammalian-adaptation characteristics of H2N2 AIVs provide a reference for early-warning and prevention for this subtype. The authors identify one emerging H2N2 AIV, reveal the molecular basis of human-type receptor binding of the H2N2 AIV and pandemic strains, and evaluate the public risk of H2N2 AIV by its infection, transmission and mammalian-adaptation features.

The game between therapeutic monoclonal antibodies (mAbs) and continuously emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has favored the virus, as most therapeutic mAbs have been evaded. Addressing this challenge, we systematically explored a reproducible bispecific antibody (bsAb)-dependent synergistic effect in this study. It could effectively restore the neutralizing activity of the bsAb when any of its single mAbs is escaped by variants. This synergy is primarily attributed to the binding angle of receptor-binding domain (RBD)-5, facilitating inter-spike cross-linking and promoting cryptic epitope exposure that classical antibody cocktails cannot achieve. Furthermore, RBD-5 with RBD-2, RBD-6, and RBD-7, alongside RBD-8, also exhibit significantly enhanced effects. This study not only shifts the paradigm in understanding antibody interactions but paves the way for developing more effective therapeutic antibodies against rapidly mutating SARS-CoV-2, with Dia-19 already showing promise against emerging variants like BA.2.86, EG.5.1, and JN.1.

Multiple SARS-CoV-2 Omicron sub-variants, such as BA.2, BA.2.12.1, BA.4, and BA.5, emerge one after another. BA.5 has become the dominant strain worldwide. Additionally, BA.2.75 is significantly increasing in some countries. Exploring their receptor binding and interspecies transmission risk is urgently needed. Herein, we examine the binding capacities of human and other 28 animal ACE2 orthologs covering nine orders towards S proteins of these sub-variants. The binding affinities between hACE2 and these sub-variants remain in the range as that of previous variants of concerns (VOCs) or interests (VOIs). Notably, R493Q reverse mutation enhances the bindings towards ACE2s from humans and many animals closely related to human life, suggesting an increased risk of cross-species transmission. Structures of S/hACE2 or RBD/hACE2 complexes for these sub-variants and BA.2S binding to ACE2 of mouse, rat or golden hamster are determined to reveal the molecular basis for receptor binding and broader interspecies recognition.

Leptin receptor (LepR) signaling plays an essential role in balancing food intake and energy expenditure. The architecture of LepR signaling assembly is critical for its function. In this study, we determined the structures of three distinct conformations of human leptin–LepR using cryo-electron microscopy at resolutions of 3.88, 3.77, and 3.58 Å. Both 2:2 and 3:3 stoichiometric assemblies were observed, and the complexes exhibited asymmetric open conformations. Leptin undergoes substantial rearrangement of its flexible regions to accommodate binding to LepR. The assembled leptin–LepR complexes connect through a “hand-in-hand” geometry. The open, interlocked 3:3 trimeric assembly results from the engagement of a third leptin–LepR heterodimer with a 2:2 dimer. The asymmetric geometry of LepR is substantially distinct from that of other gp130 cytokine homologs, and that may be due to the twisted and rigid interface between the D3 and D4 domains. These results highlight the distinct engagement of leptin with LepR and provide important insights into the structural plasticity of LepR-signaling assemblies.

The World Health Organization declared mpox (or monkeypox) a public health emergency of international concern in July 2022, and prophylactic and therapeutic measures are in urgent need. The monkeypox virus (MPXV) has its own DNA polymerase F8, together with the processive cofactors A22 and E4, constituting the polymerase holoenzyme for genome replication. Here, we determined the holoenzyme structure in complex with DNA using cryo–electron microscopy at the global resolution of ~2.8 angstroms. The holoenzyme possesses an architecture that suggests a “forward sliding clamp” processivity mechanism for viral DNA replication. MPXV polymerase has a DNA binding mode similar to that of other B-family DNA polymerases from different species. These findings reveal the mechanism of the MPXV genome replication and may guide the development of anti-poxvirus drugs.

2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and trig-gers 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 as-sembly 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 under-standing of the cGAS-STING pathway.

Highly contagious respiratory illnesses like influenza and COVID-19 pose serious risks to public health. A two-in-one vaccine would be ideal to avoid multiple vaccinations for these diseases. Here, we generated a chimeric receptor binding domain of the spike protein (S-RBD) and hemagglutinin (HA)-stalk-based vaccine for both SARS-CoV-2 and influenza viruses. The S-RBD from SARS-CoV-2 Delta was fused to the headless HA from H1N1 (H1Delta), creating a chimera that forms trimers in solution. The cryo-electron microscopy structure of the chimeric protein complexed with the RBD-targeting CB6 and the HA-stalk-targeting CR9114 antibodies shows that the trimeric protein is stable and accessible for neutralizing antibody binding. Immunization with the vaccine elicited high and long-lasting neutralizing antibodies and effectively protected mice against the challenges of lethal H1N1 or heterosubtypic H5N8, as well as the SARS-CoV-2 Delta or Omicron BA.2 variants. Overall, this study offers a two-in-one universal vaccine design to combat infections caused by both SARS-CoV-2 variants of concern and influenza viruses.

Coronaviruses(CoVs)are zoonotic pathogens that mainly cause respiratory infectious diseases and seven CoVs have been reported to infect humans[1];these viruses include the severe acute respi-ratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of coronavirus disease 2019(COVID-19).Since its identification,SARS-CoV-2 has continued to evolve,and multiple variants have emerged.

Omicron SARS-CoV-2 is rapidly spreading worldwide. To delineate the impact of emerging mutations on spike's properties, we performed systematic structural analyses on apo Omicron spike and its complexes with human ACE2 or S309 neutralizing antibody (NAb) by cryo-EM. The Omicron spike preferentially adopts the one-RBD-up conformation both before and after ACE2 binding, which is in sharp contrast to the orchestrated conformational changes to create more up-RBDs upon ACE2 binding as observed in the prototype and other four variants of concern (VOCs). Furthermore, we found that S371L, S373P and S375F substitutions enhance the stability of the one-RBD-up conformation to prevent exposing more up-RBDs triggered by ACE2 binding. The increased stability of the one-RBD-up conformation restricts the accessibility of S304 NAb, which targets a cryptic epitope in the closed conformation, thus facilitating the immune evasion by Omicron. These results expand our understanding of Omicron spike's conformation, receptor binding and antibody evasion mechanism.