Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor.

Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Herein, we present a 2.2 Å resolution X-ray crystal structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles and infectious virus. The Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement. Mxra8 binds by wedging into a cleft created by two adjacent CHIKV E2-E1 heterodimers in one trimeric spike and engaging a neighboring spike. Two binding modes are observed with the fully mature VLP, with one Mxra8 binding with unique contacts. Only the high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding-site usage. Our studies provide insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

A Molecular Code for Identity in the Vomeronasal System.

In social interactions among mammals, individuals are recognized by olfactory cues, but identifying the key signals among thousands of compounds remains a major challenge. To address this need, we developed a new technique, component-activity matching (CAM), to select candidate ligands that "explain" patterns of bioactivity across diverse complex mixtures. Using mouse urine from eight different sexes and strains, we identified 23 components to explain firing rates in seven of eight functional classes of vomeronasal sensory neurons. Focusing on a class of neurons selective for females, we identified a novel family of vomeronasal ligands, steroid carboxylic acids. These ligands accounted for much of the neuronal activity of urine from some female strains, were necessary for normal levels of male investigatory behavior of female scents, and were sufficient to trigger mounting behavior. CAM represents the first step toward an exhaustive characterization of the molecular cues for natural behavior in a mammalian olfactory system.

Human IFIT3 Modulates IFIT1 RNA Binding Specificity and Protein Stability.

Although interferon-induced proteins with tetratricopeptide repeats (IFIT proteins) inhibit infection of many viruses by recognizing their RNA, the regulatory mechanisms involved remain unclear. Here we report a crystal structure of cap 0 (m7GpppN) RNA bound to human IFIT1 in complex with the C-terminal domain of human IFIT3. Structural, biochemical, and genetic studies suggest that IFIT3 binding to IFIT1 has dual regulatory functions: (1) extending the half-life of IFIT1 and thereby increasing its steady-state amounts in cells; and (2) allosterically regulating the IFIT1 RNA-binding channel, thereby enhancing the specificity of recognition for cap 0 but not cap 1 (m7GpppNm) or 5'-ppp RNA. Mouse Ifit3 lacks this key C-terminal domain and does not bind mouse Ifit1. The IFIT3 interaction with IFIT1 is important for restricting infection of viruses lacking 2'-O methylation in their RNA cap structures. Our experiments establish differences in the regulation of IFIT1 orthologs and define targets for modulation of human IFIT protein activity.

MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM.

In Drosophila testis, myosin VI plays a special role, distinct from its motor function, by anchoring components to the unusual actin-based structures (cones) that are required for spermatid individualization. For this, the two calmodulin (CaM) light-chain molecules of myosin VI are replaced by androcam (ACaM), a related protein with 67% identity to CaM. Although ACaM has a similar bi-lobed structure to CaM, with two EF hand-type Ca2+ binding sites per lobe, only one functional Ca2+ binding site operates in the amino-terminus. To understand this light chain substitution, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to examine dynamic changes in ACaM and CaM upon Ca2+ binding and interaction with the two CaM binding motifs of myosin VI (insert2 and IQ motif). HDX-MS reveals that binding of Ca2+ to ACaM destabilizes its N-lobe but stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilization throughout. The conformation of this stable holo-C-lobe of ACaM seems to be a "prefigured" version of the conformation adopted by the holo-C-lobe of CaM for binding to insert2 and the IQ motif of myosin VI. Strikingly, the interaction of holo-ACaM with either peptide converts the holo-N-lobe to its Ca2+-free, more stable, form. Thus, ACaM in vivo should bind the myosin VI light chain sites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motor assembly and activity. These findings indicate that inhibition of myosin VI motor activity is a precondition for transition to an anchoring function.

Open conformation of tetraspanins shapes interaction partner networks on cell membranes.

Tetraspanins, including CD53 and CD81, regulate a multitude of cellular processes through organizing an interaction network on cell membranes. Here, we report the crystal structure of CD53 in an open conformation poised for partner interaction. The large extracellular domain (EC2) of CD53 protrudes away from the membrane surface and exposes a variable region, which is identified by hydrogen-deuterium exchange as the common interface for CD53 and CD81 to bind partners. The EC2 orientation in CD53 is supported by an extracellular loop (EC1). At the closed conformation of CD81, however, EC2 disengages from EC1 and rotates toward the membrane, thereby preventing partner interaction. Structural simulation shows that EC1-EC2 interaction also supports the open conformation of CD81. Disrupting this interaction in CD81 impairs the accurate glycosylation of its CD19 partner, the target for leukemia immunotherapies. Moreover, EC1 mutations in CD53 prevent the chemotaxis of pre-B cells toward a chemokine that supports B-cell trafficking and homing within the bone marrow, a major CD53 function identified here. Overall, an open conformation is required for tetraspanin-partner interactions to support myriad cellular processes.

Evaluating Chemical Footprinting-Induced Perturbation of Protein Higher Order Structure.

Specific amino acid footprinting mass spectrometry (MS) is an increasingly utilized method for elucidating protein higher order structure (HOS). It does this by adding to certain amino acid residues a mass tag, whose reaction extent depends on solvent accessibility and microenvironment of the protein. Unlike reactive free radicals and carbenes, these specific footprinters react slower than protein unfolding. Thus, their footprinting, under certain conditions, provokes structural changes to the protein, leading to labeling on non-native structures. It is critical to establish conditions (i.e., reagent concentrations, time of reaction) to ensure that the structure of the protein following footprinting remains native. Here, we compare the efficacy of five methods in assessing protein HOS following footprinting at the intact protein level and then further localize the perturbation at the peptide level. Three are MS-based methods that provide dose-response plot analysis, evaluation of Poisson distributions of precursor and products, and determination of the average number of modifications. These MS-based methods reliably and effectively indicate HOS perturbation at the intact protein level, whereas spectroscopic methods (circular dichroism (CD) and dynamic light scattering (DLS)) are less sensitive in monitoring subtle HOS perturbation caused by footprinting. Evaluation of HOS at the peptide level indicates regions that are sensitive to localized perturbations. Peptide-level analysis also provides higher resolution of the HOS perturbation, and we recommend using it for future footprinting studies. Overall, this work shows conclusive evidence for HOS perturbation caused by footprinting. Implementation of quality control workflows can identify conditions to avoid the perturbation, for footprinting, allowing accurate and reliable identification of protein structural changes that accompany, for example, ligand interactions, mutations, and changes in solution environment.

Development of a High-Throughput Mass Spectrometry-Based SARS-CoV-2 Immunoassay.

The serious impact of the Covid-19 pandemic underscores the need for rapid, reliable, and high-throughput diagnosis methods for infection. Current analytical methods, either point-of-care or centralized detection, are not able to satisfy the requirements of patient-friendly testing, high demand, and reliability of results. Here, we propose a two-point separation on-demand diagnostic strategy that uses laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) and adopts a stable yet cleavable ionic probe as a mass reporter. The use of this reporter enables ultrasensitive, interruptible, storable, restorable, and high-throughput on-demand detection. We describe a demonstration of the concept whereby we (i) design and synthesize a laser-cleavable reporter (DTPA), (ii) conjugate the reporter onto an antibody and verify the function of the conjugate, (iii) detect with good turnaround and high sensitivity the conjugated reporter, (iv) analyze quantitatively by using a laser-cleavable internal standard, and (v) identify negative and positive samples containing the spike protein. The protocol has excellent sensitivity (amol for the SARS-CoV-2 Spike S1 subunit antibody) without any amplification. This strategy is also applicable for the detection of other disease antigens besides SARS-CoV-2.

The entropic force generated by intrinsically disordered segments tunes protein function.

Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.

Medical Sanctions Against Russia: Arresting Aggression or Abrogating Healthcare Rights?

Since 2022, the EU, US, and other nations have imposed medical sanctions on Russia to block the export of pharmaceuticals and medical devices and curtail clinical trials to degrade Russia's military capabilities. While international law proscribes sanctions that cause a humanitarian crisis, an outcome averted in Russia, the military effects of medical sanctions have been lean. Strengthening medical sanctions risks violating noncombatant and combatant rights to healthcare. Each group's claim is different. Noncombatants and severely injured soldiers who cannot return to duty enjoy the right to adequate health care that sanctions cannot undermine. Combatants falling captive enjoy the same medical care that adversaries provide their own troops. Combatants yet to renounce hostilities, however, have no claim to medical attention and remain subject to sanctions. Nevertheless, medical sanctions prove unworkable in this complex environment of conflicting rights and command no place in a sustainable sanction regime.

Treating the innocent victims of trolleys and war.

Both trolleys and war leave innocent victims to suffer death and injury. Trolley problems accounting for the injured, and not only the dead, tease out intuitions about liability that enhance our understanding of the obligation to provide compensation and medical care to civilian victims of war. Like many trolley victims, civilians in war may suffer justifiable, excusable, or negligent harms that demand compensation. Chief among these is collateral harm befalling civilians. Collateral harm is endemic to war and comprises permissible but unavoidable death or injury following necessary and proportionate military operations. Although state armies sometimes offer condolence payments for civilian death, injury, and property loss, they deny liability. Instead, they use compensation to enhance counterinsurgency efforts and assuage feelings of agent regret. As part of the medical rules of eligibility, Coalition forces in Iraq and Afghanistan also provided medical care to victims of collateral harm. However, they denied care to similarly sick or injured civilians. While compensation is often justified to cure the harm civilians suffer, the differential use of medical resources is not. Rather, medical care remains subject to the principle of beneficence and medical need. The duty to provide civilian healthcare in war, particularly in wars of humanitarian intervention, is far-reaching and imposes significant costs that military and medical ethics are yet to recognize.

Structural basis for IFN antagonism by human respiratory syncytial virus nonstructural protein 2.

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNß messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.

Biochemical and HDX Mass Spectral Characterization of the SARS-CoV-2 Nsp1 Protein.

A major challenge in defining the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to better understand virally encoded multifunctional proteins and their interactions with host factors. Among the many proteins encoded by the positive-sense, single-stranded RNA genome, nonstructural protein 1 (Nsp1) stands out due to its impact on several stages of the viral replication cycle. Nsp1 is the major virulence factor that inhibits mRNA translation. Nsp1 also promotes host mRNA cleavage to modulate host and viral protein expression and to suppress host immune functions. To better define how this multifunctional protein can facilitate distinct functions, we characterize SARS-CoV-2 Nsp1 by using a combination of biophysical techniques, including light scattering, circular dichroism, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and temperature-dependent HDX-MS. Our results reveal that the SARS-CoV-2 Nsp1 N- and C-terminus are unstructured in solution, and in the absence of other proteins, the C-terminus has an increased propensity to adopt a helical conformation. In addition, our data indicate that a short helix exists near the C-terminus and adjoins the region that binds the ribosome. Together, these findings provide insights into the dynamic nature of Nsp1 that impacts its functions during infection. Furthermore, our results will inform efforts to understand SARS-CoV-2 infection and antiviral development.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

Workflow for Validating Specific Amino Acid Footprinting Reagents for Protein Higher Order Structure Elucidation.

Protein footprinting mass spectrometry probes protein higher order structure and dynamics by labeling amino acid side-chains or backbone amides as a function of solvent accessibility. One category of footprinting uses residue-specific, irreversible covalent modifications, affording flexibility of sample processing for bottom-up analysis. Although several specific amino acid footprinting technologies are becoming established in structural proteomics, there remains a need to assess fundamental properties of new reagents before their application. Often, footprinting reagents are applied to complex or novel protein systems soon after their discovery and sometimes without a thorough investigation of potential downsides of the reagent. In this work, we assemble and test a validation workflow that utilizes cyclic peptides and a model protein to characterize benzoyl fluoride, a recently published, next-generation nucleophile footprinter. The workflow includes the characterization of potential side-chain reactive groups, reaction "quench" efficacies, reagent considerations and caveats (e.g., buffer pH), residue-specific kinetics compared to those of established reagents, and protein-wide characterization of modification sites with considerations for proteolysis. The proposed workflow serves as a starting point for improved footprinting reagent discovery, validation, and introduction, the aspects of which we recommend before applying to unknown protein systems.

Characterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.

Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.