Monoclonal antibodies against S2 subunit of spike protein exhibit broad reactivity toward SARS-CoV-2 variants.

BACKGROUND: The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harbor diverse spike (S) protein sequences, which can greatly influence the efficacies of therapeutics. Therefore, it would be of great value to develop neutralizing monoclonal antibodies (mAbs) that can broadly recognize multiple variants. METHODS: Using an mRNA-LNP immunization strategy, we generated several mAbs that specifically target the conserved S2 subunit of SARS-CoV-2 (B-S2-mAbs). These mAbs were assessed for their neutralizing activity with pseudotyped viruses and binding ability for SARS-CoV-2 variants. RESULTS: Among these mAbs, five exhibited strong neutralizing ability toward the Gamma variant and also recognized viral S proteins from the Wuhan, Alpha, Beta, Gamma, Delta and Omicron (BA.1, BA.2 and BA.5) variants. Furthermore, we demonstrated the broad reactivities of these B-S2-mAbs in several different applications, including immunosorbent, immunofluorescence and immunoblotting assays. In particular, B-S2-mAb-2 exhibited potent neutralization of Gamma variant (IC50 = 0.048 µg/ml) in a pseudovirus neutralization assay. The neutralizing epitope of B-S2-mAb-2 was identified by phage display as amino acid residues 1146-1152 (DSFKEEL) in the S2 subunit HR2 domain of SARS-CoV-2. CONCLUSION: Since there are not many mAbs that can bind the S2 subunit of SARS-CoV-2 variants, our set of B-S2-mAbs may provide important materials for basic research and potential clinical applications. Importantly, our study results demonstrate that the viral S2 subunit can be targeted for the production of cross-reactive antibodies, which may be used for coronavirus detection and neutralization.

Miro1 functions as an inhibitory regulator of MFN at elevated mitochondrial Ca2+ levels.

Mitochondria function as an integrated network that moves along the microtubules within cells and changes the morphology through membrane fusion and fission events. Mitofusin (MFN) mediates membrane tethering and subsequent fusion of the mitochondrial outer membrane. Understanding the regulatory mechanisms of MFN function is critical to tackling the pathology related to mitochondrial network imbalance. Here, we reveal a novel inhibitory mechanism of MFN-mediated fusion by mitochondrial Rho GTPase (Miro1) in response to elevated mitochondrial Ca2+ concentration ([Ca2+ ]m ). We showed that elevated [Ca2+ ]m prevents the fusion between mitochondria forming the outer membrane tether by ectopically expressing MFN. Lowering [Ca2+ ]m by treating cells with an inhibitor of mitochondrial calcium uniporter or knocking down Miro1/2 induces more fused networks. Miro1 interacts with MFN as supported by co-immunoprecipitation and protein association identified by proximity labeling proteomics. It suggests that Miro1 functions as a Ca2+ -sensor and inhibits MFN function at elevated [Ca2+ ]m. Miro1 EF-hand mutant has a compromised inhibitory effect, which reiterates Ca2+ -modulated regulation. Dysregulated Ca2+ -handling and mitochondrial network imbalance are highly relevant in the pathology of cancers, cardiovascular, and neurodegenerative diseases. Miro1 functions as a coordinated Ca2+ -responder by pausing mitochondrial transport while reducing network fusion and cooperating with Drp1-mediated fission. It likely prevents the detrimental effect of Ca2+m overload and facilitates mitophagy. Our finding reveals a novel regulation of mitochondrial network dynamics responding to [Ca2+ ]m through the interplay of Miro1 and MFN. Modulation of Miro1 and MFN interaction is a potential intervention to promote network homeostasis.

Dynamic changes in mitochondrial 3D structure during folliculogenesis and luteal formation in the goat large luteal cell lineage.

In mammalian ovaries, mitochondria are integral sites of energy production and steroidogenesis. While shifts in cellular activities and steroidogenesis are well characterized during the differentiation of large luteal cells in folliculogenesis and luteal formation, mitochondrial dynamics during this process have not been previously evaluated. In this study, we collected ovaries containing primordial follicles, mature follicles, corpus hemorrhagicum, or corpus luteum from goats at specific times in the estrous cycle. Enzyme histochemistry, ultrastructural observations, and 3D structural analysis of serial sections of mitochondria revealed that branched mitochondrial networks were predominant in follicles, while spherical and tubular mitochondria were typical in large luteal cells. Furthermore, the average mitochondrial diameter and volume increased from folliculogenesis to luteal formation. In primordial follicles, the signals of cytochrome c oxidase and ATP synthase were undetectable in most cells, and the large luteal cells from the corpus hemorrhagicum also showed low enzyme signals and content when compared with granulosa cells in mature follicles or large luteal cells from the corpus luteum. Our findings suggest that the mitochondrial enlargement could be an event during folliculogenesis and luteal formation, while the modulation of mitochondrial morphology and respiratory enzyme expressions may be related to tissue remodeling during luteal formation.

Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila.

Mitochondrial DNA (mtDNA) encodes gene products that are essential for oxidative phosphorylation. They organize as higher order nucleoid structures (mtNucleoids) that were shown to be critical for the maintenance of mtDNA stability and integrity. While mtNucleoid structures are associated with cellular health, how they change in situ under physiological maturation and aging requires further investigation. In this study, we investigated the mtNucleoid assembly at an ultrastructural level in situ using the TFAM-Apex2 Drosophila model. We found that smaller and more compact TFAM-nucleoids are populated in the mitochondria of indirect flight muscle of aged flies. Furthermore, mtDNA transcription and replication were cross-regulated in the mtTFB2-knockdown flies as in the mtRNAPol-knockdown flies that resulted in reductions in mtDNA copy numbers and nucleoid-associated TFAM. Overall, our study reveals that the modulation of TFAM-nucleoid structure under physiological aging, which is critically regulated by mtDNA content.

Drosophila MICOS knockdown impairs mitochondrial structure and function and promotes mitophagy in muscle tissue.

The mitochondrial contact site and cristae organizing system (MICOS) is a multi-protein interaction hub that helps define mitochondrial ultrastructure. While the functional importance of MICOS is mostly characterized in yeast and mammalian cells in culture, the contributions of MICOS to tissue homeostasis in vivo remain further elucidation. In this study, we examined how knocking down expression of Drosophila MICOS genes affects mitochondrial function and muscle tissue homeostasis. We found that CG5903/MIC26-MIC27 colocalizes and functions with Mitofilin/MIC60 and QIL1/MIC13 as a Drosophila MICOS component; knocking down expression of any of these three genes predictably altered mitochondrial morphology, causing loss of cristae junctions, and disruption of cristae packing. Furthermore, the knockdown flies exhibited low mitochondrial membrane potential, fusion/fission imbalances, increased mitophagy, and limited cell death. Reductions in climbing ability indicated deficits in muscle function. Knocking down MICOS genes also caused reduced mtDNA content and fragmented mitochondrial nucleoid structure in Drosophila Together, our data demonstrate an essential role of Drosophila MICOS in maintaining proper homeostasis of mitochondrial structure and function to promote the function of muscle tissue.

Virus-Like Particles (VLPs) as a Platform for Hierarchical Compartmentalization.

Hierarchically self-assembled structures are common in biology, but it is often challenging to design and fabricate synthetic analogs. The archetypal cell is defined by hierarchically organized multicompartmentalized structures with boundaries that delineate the interior from exterior environments and is an inspiration for complex functional materials. Here, we have demonstrated an approach to the design and construction of a nested protein cage system that can additionally incorporate the packing of other functional macromolecules and exhibit some of the features of a minimal synthetic cell-like material. We have demonstrated a strategy for controlled co-packaging of subcompartments, ferritin (Fn) cages, together with active cellobiose-hydrolyzing ß-glycosidase enzyme macromolecules, CelB, inside the sequestered volume of the bacteriophage P22 capsid. Using controlled in vitro assembly, we were able to modulate the stoichiometry of Fn cages and CelB encapsulated inside the P22 to control the degree of compartmentalization. The co-encapsulated enzyme CelB showed catalytic activity even when packaged at high total macromolecular concentrations comparable to an intracellular environment. This approach could be used as a model to create synthetic protein-based protocells that can confine smaller functionalized proto-organelles and additional macromolecules to support a range of biochemical reactions.

Coordinated organization of mitochondrial lamellar cristae and gain of COX function during mitochondrial maturation in Drosophila.

Mitochondrial cristae contain electron transport chain complexes and are distinct from the inner boundary membrane (IBM). While many details regarding the regulation of mitochondrial structure are known, the relationship between cristae structure and function during organelle development is not fully described. Here, we used serial-section tomography to characterize the formation of lamellar cristae in immature mitochondria during a period of dramatic mitochondrial development that occurs after Drosophila emergence as an adult. We found that the formation of lamellar cristae was associated with the gain of cytochrome c oxidase (COX) function, and the COX subunit, COX4, was localized predominantly to organized lamellar cristae. Interestingly, 3D tomography showed some COX-positive lamellar cristae were not connected to IBM. We hypothesize that some lamellar cristae may be organized by a vesicle germination process in the matrix, in addition to invagination of IBM. OXA1 protein, which mediates membrane insertion of COX proteins, was also localized to cristae and reticular structures isolated in the matrix additional to the IBM, suggesting that it may participate in the formation of vesicle germination-derived cristae. Overall, our study elaborates on how cristae morphogenesis and functional maturation are intricately associated. Our data support the vesicle germination and membrane invagination models of cristae formation.

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography.

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

Generation and Characterization of Antinonstructural Protein 1 Monoclonal Antibodies and Development of Diagnostics for Dengue Virus Serotype 2.

Dengue virus (DENV) circulates in tropical and subtropical areas around the world, where it causes high morbidity and mortality. There is no effective treatment of infection, with supportive care being the only option. Furthermore, early detection and diagnosis are important to facilitate clinical decisions. In this study, seven monoclonal antibodies (mAbs) recognizing nonstructural protein 1 (NS1) of DENV were generated by hybridoma techniques. These antibodies can be divided into two groups: serotype-specific (DB6-1, DB12-3, and DB38-1) and nonspecific (consisting of antibodies DB16-1, DB20-6, DB29-1, and DB41-2). The B-cell epitopes of DB20-6 and DB29-1 were identified by phage display and site-directed mutagenesis, and its binding motif, WXXWGK, was revealed to correspond to amino acid residues 115-120 of the DENV-2 NS1 protein. A diagnostic platform, consisting of a serotype-specific capture antibody and a complex detection antibody, exhibited a detection limit of about 1 ng/mL, which is sufficient to detect NS1 in clinical serum samples from dengue patients. This diagnostic platform displayed better specificity and sensitivity than two examined commercial NS1 diagnostic platforms. In summary, our results indicate that these newly generated mAbs are suitable for detection of NS1 protein of DENV-2 in clinical samples.

Electron tomographic analysis reveals ultrastructural features of mitochondrial cristae architecture which reflect energetic state and aging.

Within mitochondria, the ability to produce energy relies upon the architectural hallmarks of double membranes and cristae invaginations. Herein, we describe novel features of mitochondrial cristae structure, which correspond to the energetic state of the organelle. In concordance with high-energy demand, mitochondria of Drosophila indirect flight muscle exhibited extensive intra-mitochondrial membrane switches between densely packed lamellar cristae that resulted in a spiral-like cristae network and allowed for bidirectional matrix confluency. This highly interconnected architecture is expected to allow rapid equilibration of membrane potential and biomolecules across integrated regions. In addition, mutant flies with mtDNA replication defect and an accelerated aging phenotype accumulated mitochondria that contained subsections of swirling membrane alongside normal cristae. The swirling membrane had impaired energy production capacity as measured by protein composition and function. Furthermore, mitochondrial fusion and fission dynamics were affected in the prematurely aged flies. Interestingly, the normal cristae that remained in the mitochondria with swirling membranes maintained acceptable function that camouflaged them from quality control elimination. Overall, structural features of mitochondrial cristae were described in three-dimension from serial section electron tomographic analysis which reflect energetic state and mtDNA-mediated aging.

High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.

Maximizing the potential of electron cryomicroscopy data collected using direct detectors.

Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without conversion into light) and feature significantly improved detective quantum efficiencies and readout rates as compared to photographic films or CCDs. We evaluated here the potential of one such detector (Gatan K2 Summit) to enable the achievement of near-atomic resolution reconstructions of biological specimens when coupled to a widely used, mid-range transmission electron microscope (FEI TF20 Twin). Compensating for beam-induced motion and stage drift provided a 4.4Å resolution map of Sulfolobus turreted icosahedral virus (STIV), which we used as a test particle in this study. Several motion correction and dose fractionation procedures were explored and we describe their influence on the resolution of the final reconstruction. We also compared the quality of this data to that collected with a FEI Titan Krios microscope equipped with a Falcon I direct detector, which provides a benchmark for data collected using a high-end electron microscope.

Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography.

Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (ß-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution.

Structure and cell biology of archaeal virus STIV.

Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interactions. Reliable laboratory conditions to propagate STIV and available genetic tools allowed structural characterization of the virus and viral components that lead to the proposal of common capsid ancestry with PRD1 (bacteriophage), Adenovirus (eukaryotic virus) and PBCV (chlorellavirus). Microarray and proteomics approaches systematically analyzed viral replication and the corresponding host responses. Cellular cryo-electron tomography and thin-section EM studies uncovered the assembly and maturation pathway of STIV and revealed dramatic cellular ultra-structure changes upon infection. The viral-induced pyramid-like protrusions on cell surfaces represent a novel viral release mechanism and previously uncharacterized functions in viral replication.

Viral life cycles captured in three-dimensions with electron microscopy tomography.

Viruses hijack host cell functions and optimize them for viral replication causing a severe threat to human health. However, viruses are also tools to understand cell biology and they may be effective reagents in nano-medicine. Studies from the molecular to cellular levels are aimed at understanding the details of viral life cycles and the underlying virus-host interactions. Recent developments in electron microscopy tomography allow viral and cellular events to be observed in fine structural detail in three-dimension. By combining high-resolution structures of individual proteins and macro-complexes obtained by crystallography and electron cryo-microscopy and image reconstruction with reconstructions performed on sub-tomographic volumes, electron tomography has advanced the structural and mechanistic understanding of virus infections both in vitro and in host cells.

Streptococcal M1 protein constructs a pathological host fibrinogen network.

M1 protein, a major virulence factor of the leading invasive strain of group A Streptococcus, is sufficient to induce toxic-shock-like vascular leakage and tissue injury. These events are triggered by the formation of a complex between M1 and fibrinogen that, unlike M1 or fibrinogen alone, leads to neutrophil activation. Here we provide a structural explanation for the pathological properties of the complex formed between streptococcal M1 and human fibrinogen. A conformationally dynamic coiled-coil dimer of M1 was found to organize four fibrinogen molecules into a specific cross-like pattern. This pattern supported the construction of a supramolecular network that was required for neutrophil activation but was distinct from a fibrin clot. Disruption of this network into other supramolecular assemblies was not tolerated. These results have bearing on the pathophysiology of streptococcal toxic shock.

In vivo virus structures: simultaneous classification, resolution enhancement, and noise reduction in whole-cell electron tomography.

Sulfolobus Turreted Icosahedral Virus (STIV) experiences an extra-cellular environment of near boiling acid (80°C, pH 3) and particles purified under these conditions were previously analyzed by cryo electron microscopy and image reconstruction. Here we describe cryo-tomograms of Solfolobus cells infected with STIV and the maximum likelihood algorithm employed to compute reconstructions of virions within the cell. Virions in four different tomograms were independently reconstructed with an average of 91 particles per tomogram and their structures compared with each other and with the higher resolution single-particle reconstruction from purified virions. The algorithm described here automatically classified and oriented two different particle types within each cell and generated reconstructions of full and empty particles. Because the particles are randomly oriented within the cell, the reconstructions do not suffer from the missing wedge of data absent from the reciprocal-space tomogram. The fact that the particles have icosahedral symmetry is used to dramatically improve the signal to noise ratio in the reconstructions. The reconstructions have approximately 60Å resolution (based on Fourier Shell Correlation analysis among reconstructions computed by the algorithm described here from four different tomograms).

Transferrin-mediated targeting of bacteriophage HK97 nanoparticles into tumor cells.

AIMS: Next-generation targeted nanodevices are currently under development for imaging and therapeutic applications. We engineered HK97 viral nanoparticles (VNPs) for tumor cell-specific targeting. METHODS: A combination of genetic and chemical engineering methods were developed and applied to generate dual-labeled HK97 cysteine mutant particles displaying transferrin and fluorescent labels. The targeting properties of transferrin-conjugated VNPs were evaluated by in vitro experiments using different cancer cell lines. RESULTS: We found that HK97-transferrin formulations were indeed targeted to cancer cells in vitro via the transferrin receptor. These studies highlight the utility and facilitate the further development of HK97-based VNPs.

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography.

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.

The architecture and chemical stability of the archaeal Sulfolobus turreted icosahedral virus.

Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-A-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-A resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.