ABSTRACT
Objective: To investigate the short-term effectiveness of unilateral biportal endoscopy (UBE) in treatment of lumbar lateral saphenous fossa combined with intervertebral foramina stenosis via contralateral sublaminar approach. Methods: A clinical data of 15 patients with lumbar lateral saphenous fossa combined with intervertebral foramina stenosis, who were admitted between September 2021 and December 2023 and met selective criteria, was retrospectively analyzed. There were 5 males and 10 females with an average age of 70.3 years (range, 46-83 years). Surgical segment was L 4, 5 in 12 cases and L 5, S 1 in 3 cases. The disease duration was 12-30 months (mean, 18.7 months). All patients were treated by UBE via contralateral sublaminar approach. The operation time, intraoperative blood loss, postoperative hospital stay, and the occurrence of complications were recorded. The visual analogue scale (VAS) score was used to evaluate the degree of lower back and leg pain before and after operation; the Japanese Orthopaedic Association (JOA) score and the Oswestry disability index (ODI) were used to evaluate the lumbar function; and the clinical outcome was evaluated using the MacNab criteria at 6 months after operation. Postoperative MRI and CT were taken to observe whether the lateral saphenous fossa and intervertebral foramen stenosis were removed or not, and the cross-sectional area of the spinal canal (CSA-SC), cross-sectional area of the intervertebral foramen (CSA-IVF), and cross-sectional area of the facet joint (CSA-FJ) were measured. Results: The operation time was 55-200 minutes (mean, 127.5 minutes); the intraoperative blood loss was 10-50 mL (mean, 27.3 mL); the length of postoperative hospital stay was 3-12 days (mean, 6.8 days). All patients were followed up 6-12 months (mean, 8.9 months). At 1 day, 1 month, 3 months, and 6 months after operation, the VAS scores of low back and leg pain and ODI scores after operation were significantly lower than preoperative scores and showed a gradual decrease with time; the JOA scores showed a gradual increase with time; the differences in the above indexes between different time points were significant ( P<0.05). The clinical outcome was rated as excellent in 10 cases, good in 4 cases, and poor in 1 case according to the MacNab criteria at 6 months after operation, with an excellent and good rate of 93.33%. Imaging review showed that the compression on the lateral saphenous fossa and intervertebral foramina had been significantly relieved, and the affected articular process joint was preserved to the maximum extent; the CSA-SC and CSA-IVF at 3 days after operation significantly increased compared to the preoperative values ( P<0.05), and the CSA-FJ significantly reduced ( P<0.05). Conclusion: The UBE via contralateral sublaminar approach can effectively reduce pressure in the lateral saphenous fossa and the intervertebral foramina of the same segment while preserving the bilateral articular process joints. The short-term effectiveness is good and it is expected to avoid fusion surgery caused by iatrogenic instability of the lumbar spine. However, further follow-up is needed to clarify the mid- and long-term effectiveness.
Subject(s)
Endoscopy , Lumbar Vertebrae , Spinal Stenosis , Humans , Male , Female , Spinal Stenosis/surgery , Middle Aged , Aged , Lumbar Vertebrae/surgery , Endoscopy/methods , Treatment Outcome , Aged, 80 and over , Decompression, Surgical/methods , Pain Measurement , Low Back Pain/etiology , Low Back Pain/surgeryABSTRACT
As SARS-CoV-2 Omicron and other variants of concern (VOCs) continue spreading worldwide, development of antibodies and vaccines to confer broad and protective activity is a global priority. Here, we report on the identification of a special group of nanobodies from immunized alpaca with potency against diverse VOCs including Omicron subvariants BA.1, BA.2 and BA.4/5, SARS-CoV-1, and major sarbecoviruses. Crystal structure analysis of one representative nanobody, 3-2A2-4, discovers a highly conserved epitope located between the cryptic and the outer face of the receptor binding domain (RBD), distinctive from the receptor ACE2 binding site. Cryo-EM and biochemical evaluation reveal that 3-2A2-4 interferes structural alteration of RBD required for ACE2 binding. Passive delivery of 3-2A2-4 protects K18-hACE2 mice from infection of authentic SARS-CoV-2 Delta and Omicron. Identification of these unique nanobodies will inform the development of next generation antibody therapies and design of pan-sarbecovirus vaccines.
Subject(s)
COVID-19 , Camelids, New World , Severe acute respiratory syndrome-related coronavirus , Single-Domain Antibodies , Animals , Mice , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , COVID-19/prevention & control , Antibodies, Neutralizing , Spike Glycoprotein, Coronavirus , Antibodies, ViralABSTRACT
Conserved and multifunctional Geminivirus Replication-associated Protein (Rep) specifically recognizes the replication origin and initiates viral DNA replication. We report the X-ray crystallography-based structures of two complexes containing the N-terminal domain (5-117aa) of Tomato yellow leaf curl virus (TYLCV) Rep: the catalytically-dead Rep in complex with nonanucleotide ssDNA (Rep5-117 Y101F-ssDNA) as well as the catalytically-active phosphotyrosine covalent adduct (Rep5-117-ssDNA). These structures provide functional insight into the role of Rep in viral replication. Metal ions stabilize the DNA conformation by interacting with the phosphate group of adenine and thus promote formation of the catalytic center. Furthermore, we identified a compound that inhibits the binding of Rep to ssDNA and dsDNA and found that the addition of metal ions compromises the inhibitory effectiveness of this compound. This study demonstrates the mechanism of DNA recognition and cleavage process of viral Rep, emphasizing the role of metal ions.
Subject(s)
Begomovirus , Solanum lycopersicum , Begomovirus/genetics , Begomovirus/metabolism , DNA Replication , DNA, Viral/genetics , DNA, Viral/metabolism , Solanum lycopersicum/genetics , Virus Replication/geneticsABSTRACT
Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.
Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Receptors, Virus/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites/immunology , COVID-19/immunology , Cricetinae , Disease Models, Animal , Epitopes/immunology , Female , Humans , Male , Neutralization Tests , Receptors, Antigen, B-Cell/immunology , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.
Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Binding Sites , COVID-19/virology , Disease Models, Animal , Epitopes , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , COVID-19 Drug TreatmentABSTRACT
PURPOSE OF REVIEW: The spike glycoprotein plays a critical role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by recognizing the angiotensin converting enzyme 2 (ACE2) receptor and mediating fusion of the viral envelope with the cell membrane. It is also the major target for neutralizing antibodies and vaccines. This review summarizes recent studies on the structure and function of spike glycoprotein, which revealed the structural basis of SARS-CoV-2 infection. RECENT FINDINGS: SARS-CoV-2 spike glycoprotein, similar to those of SARS-CoV and Middle East respiratory syndrome coronavirus, spontaneously samples different prefusion states with the receptor-binding domain (RBD) adopting 'up' or 'down' conformations, and the RBD 'down' to 'up' conformational change is required for ACE2 binding. Receptor binding and spike glycoprotein priming by host proteases such as furin and transmembrane protease serine 2 induce pre to postfusion conformational changes of the spike trimer that enable membrane fusion. Interactions between SARS-CoV-2 RBD and ACE2 were elucidated at atomic resolution using high-resolution crystal structures. These structures, together with adapted and remodeled SARS-CoV-2 strains, further revealed critical residues of the spike glycoprotein for SARS-CoV-2 infection and cross-species transmission. SUMMARY: Recent studies on SARS-CoV-2 spike glycoprotein provide important structural knowledge for a better understanding of the molecular mechanisms of SARS-CoV-2 infection and cross-species transmission.
Subject(s)
COVID-19/virology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , COVID-19/enzymology , COVID-19/genetics , Humans , Protein Binding , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/blood , Antibodies, Viral/metabolism , Antibodies, Viral/ultrastructure , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Infections/blood , Coronavirus Infections/virology , Crystallography, X-Ray , Dipeptidyl Peptidase 4/metabolism , Disease Models, Animal , Epitope Mapping , Epitopes/immunology , Female , HEK293 Cells , Humans , Mice , Middle East Respiratory Syndrome Coronavirus/metabolism , Neutralization Tests , Protein Binding/immunology , Protein Domains/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , Vero Cells , Virus InternalizationABSTRACT
The Middle East respiratory syndrome coronavirus (MERS-CoV) has spread through 27 countries and infected more than 2,200 people since its first outbreak in Saudi Arabia in 2012. The high fatality rate (35.4%) of this novel coronavirus and its persistent wide spread infectiousness in animal reservoirs have generated tremendous global public health concern. However, no licensed therapeutic agents or vaccines against MERS-CoV are currently available and only a limited few have entered clinical trials. Among all the potential targets of MERS-CoV, the spike glycoprotein (S) has been the most well-studied due to its critical role in mediating viral entry and in inducing a protective antibody response in infected individuals. The most notable studies include the recent discoveries of monoclonal antibodies and development of candidate vaccines against the S glycoprotein. Structural characterization of MERS-CoV S protein bound with these monoclonal antibodies has provided insights into the mechanisms of humoral immune responses against MERS-CoV infection. The current review aims to highlight these developments and discuss possible hurdles and strategies to translate these discoveries into ultimate medical interventions against MERS-CoV infection.
Subject(s)
Antibodies, Viral/immunology , Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/immunology , Viral Vaccines/immunology , Animals , Coronavirus Infections/immunology , Coronavirus Infections/virology , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/geneticsABSTRACT
Most neutralizing antibodies against highly pathogenic avian influenza A virus H5N1 recognize the receptor-binding site (RBS) on the globular head domain and the stem of H5N1 hemagglutinin (HA). Through comprehensive analysis of multiple human protective antibodies, we previously identified four vulnerable sites (VS1-VS4) on the globular head domain. Among them, the VS1, occupying the opposite side of the RBS on the same HA, was defined by the epitope of antibody 65C6. In this study, we report the crystal structures of two additional human H5N1 antibodies isolated from H5N1-infected individuals, 3C11 and AVFluIgG01, bound to the head at 2.33- and 2.30-Å resolution, respectively. These two new antibody epitopes have large overlap with and extend beyond the original VS1. Site-directed mutagenesis experiments identified eight pivotal residues (Ser-126b, Lys-165, Arg-166, Ser-167, Tyr-168, Asn-169, Thr-171, and Asn-172) critical for 65C6-, 3C11-, and AVFluIgG01-binding and neutralization activities. These residues formed a unique "Y"-shaped surface on H5N1 globular head and are highly conserved among H5N1 viruses. Our results further support the existence of a vulnerable site distinct from the RBS and the stem region of H5N1 HA, and future design of immunogens should take this particular site into consideration.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza A Virus, H5N1 Subtype/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites , Crystallography, X-Ray , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A Virus, H5N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/pathogenicity , Mutagenesis, Site-Directed , Protein Conformation , Reproducibility of ResultsABSTRACT
AIDS has evolved from a fatal infectious disease to a manageable chronic disease under the treatment of anti-AIDS medications. HIV fusion inhibitors with high activity, low side effects and strong selectivity are promising drugs against HIV. Only one fusion inhibitor is currently approved, thereby highly active long-acting fusion inhibitors need to be developed for long-term AIDS treatment. Here, we synthesised MT-SC22EK (a small HIV fusion inhibitor) derivatives containing 1-2 staples to improve its stability. Antiviral activity studies showed that MT-SC22EK-2 with two staples exhibited potent inhibitory activity against HIV-1 standard strains and Chinese epidemic strains, and at the same time, MT-SC22EK-2 presented strong anti-T20 resistance. Surprisingly, MT-SC22EK-2 possessed excellent protease stability with a half-life of 3665 min. MT-SC22EK-2 is a potential HIV fusion inhibitor considered as a long-acting anti-HIV drug candidate.
ABSTRACT
The major mechanism of antibody-mediated neutralization of the Middle East respiratory syndrome coronavirus (MERS-CoV) involves competition with the cellular receptor dipeptidyl peptidase 4 (DPP4) for binding to the receptor-binding domain (RBD) of the spike (S) glycoprotein. Here, we report a unique epitope and unusual neutralizing mechanism of the isolated human antibody MERS-4. Structurally, MERS-4 approached the RBD from the outside of the RBD-DPP4 binding interface. Such binding resulted in the folding of the ß5-ß6 loop toward a shallow groove on the RBD interface critical for accommodating DPP4. The key residues for binding are identified through site-directed mutagenesis. Structural modeling revealed that MERS-4 binds to RBD only in the "up" position in the S trimer. Furthermore, MERS-4 demonstrated synergy with several reported antibodies. These results indicate that MERS-4 neutralizes MERS-CoV by indirect rather than direct competition with DPP4. This mechanism provides a valuable addition for the combined use of antibodies against MERS-CoV infection.
Subject(s)
Antibodies, Neutralizing/metabolism , Epitopes/chemistry , Middle East Respiratory Syndrome Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Binding Sites , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Models, Molecular , Mutation/genetics , Protein Binding , Protein Domains , Protein MultimerizationABSTRACT
Background: The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection with a high (~35%) mortality rate. Neutralizing antibodies targeting the spike of MERS-CoV have been shown to be a therapeutic option for treatment of lethal disease. Methods: We describe the germline diversity and neutralizing activity of 13 potent human monoclonal antibodies (mAbs) that target the MERS-CoV spike (S) protein. Biological functions were assessed by live MERS-CoV, pseudotype particle and its variants, and structural basis was also determined by crystallographic analysis. Results: Of the 13 mAbs displaying strong neutralizing activity against MERS-CoV, two with the immunoglobulin heavy-chain variable region (IGHV)1-69-derived heavy chain (named MERS-GD27 and MERS-GD33) showed the most potent neutralizing activity against pseudotyped and live MERS-CoV in vitro. Mutagenesis analysis suggested that MERS-GD27 and MERS-GD33 recognized distinct regions in S glycoproteins, and the combination of 2 mAbs demonstrated a synergistic effect in neutralization against pseudotyped MERS-CoV. The structural basis of MERS-GD27 neutralization and recognition revealed that its epitope almost completely overlapped with the receptor-binding site. Conclusions: Our data provide new insights into the specific antibody repertoires and the molecular determinants of neutralization during natural MERS-CoV infection in humans. This finding supports additional efforts to design and develop novel therapies to combat MERS-CoV infections in humans.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Antibodies, Monoclonal/immunology , Antibody Specificity , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Humans , Male , Models, Molecular , Protein ConformationABSTRACT
The deep hydrophobic pocket of HIV-1 gp41 has been considered a drug target, but short-peptides targeting this site usually lack potent antiviral activity. By applying the M-T hook structure, we previously generated highly potent short-peptide fusion inhibitors that specifically targeted the pocket site, such as MT-SC22EK, HP23L, and LP-11. Here, the crystal structures of HP23L and LP-11 bound to the target mimic peptide N36 demonstrated the critical intrahelical and interhelical interactions, especially verifying that the hook-like conformation was finely adopted while the methionine residue was replaced by the oxidation-less prone residue leucine, and that addition of an extra glutamic acid significantly enhanced the binding and inhibitory activities. The structure of HP23L bound to N36 with two mutations (E49K and L57R) revealed the critical residues and motifs mediating drug resistance and provided new insights into the mechanism of action of inhibitors. Therefore, the present data help our understanding for the structure-activity relationship (SAR) of HIV-1 fusion inhibitors and facilitate the development of novel antiviral drugs.
Subject(s)
Drug Resistance, Viral , HIV Envelope Protein gp41/chemistry , HIV Fusion Inhibitors/chemistry , HIV-1 , Viral Fusion Proteins/chemistry , Amino Acid Sequence , Amino Acid Substitution , Antiviral Agents , Crystallography, X-Ray , Drug Delivery Systems , Drug Design , Glutamic Acid , HEK293 Cells , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/pharmacology , HIV Fusion Inhibitors/pharmacology , HIV-1/chemistry , HIV-1/genetics , Humans , Leucine , Models, Molecular , Mutation, Missense , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides/chemistry , Peptides/genetics , Peptides/pharmacology , Protein Conformation , Protein Domains/genetics , Protein Interaction Domains and Motifs , Retroviridae Proteins/genetics , Retroviridae Proteins/pharmacology , Structure-Activity Relationship , Viral Fusion Proteins/genetics , Viral Fusion Proteins/pharmacologyABSTRACT
Autophagic lysosome reformation (ALR) plays an important role in maintaining lysosome homeostasis. During ALR, lysosomes are reformed by recycling lysosomal components from autolysosomes. The most noticeable step of ALR is autolysosome tubulation, but it is currently unknown how the process is regulated. Here, using an approach combining in vivo studies and in vitro reconstitution, we found that the kinesin motor protein KIF5B is required for autolysosome tubulation and that KIF5B drives autolysosome tubulation by pulling on the autolysosomal membrane. Furthermore, we show that KIF5B directly interacts with PtdIns(4,5)P2. Kinesin motors are recruited and clustered on autolysosomes via interaction with PtdIns(4,5)P2 in a clathrin-dependent manner. Finally, we demonstrate that clathrin promotes formation of PtdIns(4,5)P2-enriched microdomains, which are required for clustering of KIF5B. Our study reveals a mechanism by which autolysosome tubulation was generated.
Subject(s)
Autophagy , Kinesins/metabolism , Lysosomes/metabolism , Animals , Clathrin/metabolism , Liposomes/metabolism , Membrane Microdomains/metabolism , Models, Biological , Phosphatidylinositol 4,5-Diphosphate/metabolism , RatsABSTRACT
The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans with an approximately 30% mortality rate. The envelope spike glycoprotein on the surface of MERS-CoV mediates receptor binding, membrane fusion, and viral entry. We previously reported two human monoclonal antibodies that target the receptor binding domain (RBD) of the spike and exhibit strong neutralization activity against live and pesudotyped MERS-CoV infection. Here we determined the crystal structure of MERS-CoV RBD bound to the Fab fragment of MERS-27 antibody at 3.20 Å resolution. The MERS-27 epitope in the RBD overlaps with the binding site of the MERS-CoV receptor DPP4. Further biochemical, viral entry, and neutralization analyses identified two critical residues in the RBD for both MERS-27 recognition and DPP4 binding. One of the residues, Trp535, was found to function as an anchor residue at the binding interface with MERS-27. Upon receptor binding, Trp535 interacts with the N-linked carbohydrate moiety of DPP4. Thus, MERS-27 inhibits MERS-CoV infection by directly blocking both protein-protein and protein-carbohydrate interactions between MERS-CoV RBD and DPP4. These results shed light on the molecular basis of MERS-27 neutralization and will assist in the optimization of MERS-27 as a tool to combat MERS-CoV infection.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/ultrastructure , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/ultrastructure , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/ultrastructure , Antiviral Agents/chemistry , Binding Sites , Humans , Models, Chemical , Molecular Docking Simulation , Protein Binding , Structure-Activity RelationshipABSTRACT
Mitochondria form networks. Formation of mitochondrial networks is important for maintaining mitochondrial DNA integrity and interchanging mitochondrial material, whereas disruption of the mitochondrial network affects mitochondrial functions. According to the current view, mitochondrial networks are formed by fusion of individual mitochondria. Here, we report a new mechanism for formation of mitochondrial networks through KIF5B-mediated dynamic tubulation of mitochondria. We found that KIF5B pulls thin, highly dynamic tubules out of mitochondria. Fusion of these dynamic tubules, which is mediated by mitofusins, gives rise to the mitochondrial network. We further demonstrated that dynamic tubulation and fusion is sufficient for mitochondrial network formation, by reconstituting mitochondrial networks in vitro using purified fusion-competent mitochondria, recombinant KIF5B, and polymerized microtubules. Interestingly, KIF5B only controls network formation in the peripheral zone of the cell, indicating that the mitochondrial network is divided into subzones, which may be constructed by different mechanisms. Our data not only uncover an essential mechanism for mitochondrial network formation, but also reveal that different parts of the mitochondrial network are formed by different mechanisms.
Subject(s)
Kinesins/metabolism , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Dynamics , Animals , Cell Line , GTP Phosphohydrolases , Humans , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , RatsABSTRACT
The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 Å-resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 ß-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell. The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.
Subject(s)
Coronavirus/metabolism , Dipeptidyl Peptidase 4/metabolism , Viral Proteins/metabolism , Amino Acid Substitution , Animals , Binding Sites , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/genetics , Humans , Protein Binding , Protein Structure, Tertiary , Severe acute respiratory syndrome-related coronavirus/metabolism , Sf9 Cells , Spodoptera , Viral Proteins/chemistry , Viral Proteins/genetics , Virus InternalizationABSTRACT
The R-spondin (RSPO) family of secreted proteins (RSPO1-RSPO4) has pleiotropic functions in development and stem cell growth by strongly enhancing Wnt pathway activation. Recently, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), LGR5, and LGR6 have been identified as receptors for RSPOs. Here we report the complex structure of the LGR4 extracellular domain (ECD) with the RSPO1 N-terminal fragment (RSPO1-2F) containing two adjacent furin-like cysteine-rich domains (FU-CRDs). The LGR4-ECD adopts the anticipated TLR horseshoe structure and uses its concave surface close to the N termini to bind RSPO1-2F. Both the FU-CRD1 and FU-CRD2 domains of RSPO1 contribute to LGR4 interaction, and binding and cellular assays identified critical RSPO1 residues for its biological activities. Our results define the molecular mechanism by which the LGR4/5/6 receptors recognize RSPOs and also provide structural insights into the signaling difference between the LGR4/5/6 receptors and other members in the LGR family.
Subject(s)
Models, Molecular , Thrombospondins/chemistry , Thrombospondins/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Sequence Alignment , Signal Transduction , Thrombospondins/geneticsABSTRACT
Broadly neutralizing antibodies and appropriate immunogens are critical for preexposure prophylaxis and therapeutic HIV vaccines. In this study, we aimed to explore effective antibodies against the genetically diverse HIV-1 strains by investigating the roles of human CD4 D1D2 domain and nonvariable immugens. The human CD4 D1D2 domain and the chimeric protein of mouse D1 domain/human D2 domain were expressed in Sf9 insect cells and purified by gel-filtration chromatography. The human CD4 D1D2 domain potently inhibited the infection of 77.8% HIV-1 pseudoviruses, including the clades AE, B' and BC, with less than 20 µg/mL of IC(50). pcDNA3.1-mhD1D2m and pcDNA3.1-mhD2m plasmids were used for the production of mouse anti-human CD4 polyclonal antibodies. The neutralizing activities of the polyclonal antibodies were determined by using pseudotyped HIV-1 viruses. The antibodies induced by plasmids containing human CD4 D1D2 domain were able to potently inhibit all pseudotyped HIV-1 strains. The antibodies from mhD1D2m-immunized mice also showed strong binding capacity to CD4 expressed on the surface of TZM-bl cells. The potent and broad inhibitory activity of antibodies against the human CD4 D1D2 domain may be used to develop effective passive immunization agent to control the spread of HIV infection.
Subject(s)
CD4 Antigens/chemistry , CD4 Antigens/immunology , HIV Antibodies/biosynthesis , HIV Infections/prevention & control , HIV-1/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/genetics , AIDS Vaccines/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/immunology , CD4 Antigens/genetics , CD4 Antigens/metabolism , Cell Line , Cells, Cultured , Female , HIV Antibodies/immunology , HIV Infections/immunology , Humans , Immunization , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neutralization Tests , Protein Structure, Tertiary , SpodopteraABSTRACT
VRC01, a broadly neutralizing monoclonal antibody, is capable of neutralizing a diverse array of HIV-1 isolates by mimicking CD4 binding with the envelope glycoprotein gp120. Nonetheless, resistant strains have been identified. Here, we examined two genetically related and two unrelated envelope clones, derived from CRF08_BC-infected patients, with distinct VRC01 neutralization profiles. A total of 22 chimeric envelope clones was generated by interchanging the loop D and/or V5 regions between the original envelopes or by single alanine substitutions within each region. Analysis of pseudoviruses built from these mutant envelopes showed that interchanging the V5 region between the genetically related or unrelated clones completely swapped their VRC01 sensitivity profiles. Mutagenesis analysis revealed that the asparagine residue at position 460 (Asn-460), a potential N-linked glycosylation site in the V5 region, is a key factor for observed resistance in these strains, which is further supported by our structural modeling. Moreover, changes in resistance were found to positively correlate with deviations in VRC01 binding affinity. Overall, our study indicates that Asn-460 in the V5 region is a critical determinant of sensitivity to VRC01 specifically in these viral strains. The long side chain of Asn-460, and potential glycosylation, may create steric hindrance that lowers binding affinity, thereby increasing resistance to VRC01 neutralization.