Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

The loss of LRPPRC function induces the mitochondrial unfolded protein response.

The inactivation of the LRPPRC gene, which has previously been associated with the neurodegenerative French Canadian Leigh Syndrome, results in a decrease in the production of mitochondria-encoded subunits of complex IV, thereby causing a reduction in complex IV activity. Previously we have shown that reducing complex IV activity triggers a compensatory and conserved mitochondrial hyperfusion response. We now demonstrate that LRPPRC knock-down in mammalian cells leads to an imbalance between mitochondria-encoded and nuclear-encoded subunits of complex IV and that this imbalance triggers the mitochondrial unfolded protein response (UPR(mt)). The inactivation of the LRPPRC-like gene mma-1 in C. elegans also induces UPR(mt), which demonstrates that this response is conserved. Furthermore, we provide evidence that mitochondrial hyperfusion and UPR(mt) are coordinated but mediated by genetically distinct pathways. We propose that in the context of LRPPRC mma-1 knock-down, mitochondrial hyperfusion helps to transiently maintain mitochondrial ATP production while UPR(mt) participates in the restoration of mitochondrial proteostasis. Mitochondrial proteostasis is not only critical in pathophysiology but also during aging, as proteotoxic stress has been shown to increase with age. Therefore, we speculate that the coordination of these two mitochondrial stress responses plays a more global role in mitochondrial proteostasis.

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling.

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

FoxO1 induces Ikaros splicing to promote immunoglobulin gene recombination.

Somatic rearrangement of immunoglobulin (Ig) genes is a key step during B cell development. Using pro-B cells lacking the phosphatase Pten (phosphatase and tensin homolog), which negatively regulates phosphoinositide-3-kinase (PI3K) signaling, we show that PI3K signaling inhibits Ig gene rearrangement by suppressing the expression of the transcription factor Ikaros. Further analysis revealed that the transcription factor FoxO1 is crucial for Ikaros expression and that PI3K-mediated down-regulation of FoxO1 suppresses Ikaros expression. Interestingly, FoxO1 did not influence Ikaros transcription; instead, FoxO1 is essential for proper Ikaros mRNA splicing, as FoxO1-deficient cells contain aberrantly processed Ikaros transcripts. Moreover, FoxO1-induced Ikaros expression was sufficient only for proximal V(H) to DJ(H) gene rearrangement. Simultaneous expression of the transcription factor Pax5 was needed for the activation of distal V(H) genes; however, Pax5 did not induce any Ig gene rearrangement in the absence of Ikaros. Together, our results suggest that ordered Ig gene rearrangement is regulated by distinct activities of Ikaros, which mediates proximal V(H) to DJ(H) gene rearrangement downstream of FoxO1 and cooperates with Pax5 to activate the rearrangement of distal V(H) genes.

Efficient generation of B lymphocytes by recognition of self-antigens.

Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock-in model system of the 3-83 autoreactive B-cell antigen receptor (BCR) in combination with precursor-BCR (pre-BCR) deficiency, we show here that the 3-83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto-antigen. Experiments with mixed bone marrow chimeras showed that combining the 3-83 BCR with the corresponding auto-antigen resulted in efficient reconstitution of B-cell development in immune-deficient mice. These results suggest that B cells are positively selected by recognition of self-antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre-BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains.

Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.

Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.

A co-localization assay for the analysis of protein-protein interactions.

The understanding and analysis of protein associations in living cells is a major goal of molecular biology. Here, we describe an assay for the analysis of protein-protein interactions based on the co-localization of a fused site-specific protease with a cleavable reporter in close proximity to the interaction partner under examination. We exemplified this scheme in the temperature-sensitive Saccharomyces cerevisiae cdc25-2 mutant strain using the nuclear inclusion protease of tobacco etch virus fused to the adaptor protein growth factor receptor binding protein 2 (Grb2). The growth-defective phenotype of cdc25-2 was complemented by expression of a membrane-targeted constitutively active Ras protein, which contained a TEV protease substrate sequence allowing for release from the membrane upon proteolysis. Interaction of Grb2 with the membrane-targeted intracellular domain of the oncogene vErbB resulted in co-localization of the TEV protease with its substrate, release of Ras from the membrane and restoration of the temperature-sensitive phenotype of cdc25-2. The flexibility of the general scheme of this approach may allow for its application in many different assay scenarios and may represent a suitable alternative in cases where other approaches fail.

Deregulated Syk inhibits differentiation and induces growth factor-independent proliferation of pre-B cells.

The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are then able to induce leukemia in mice. Syk-transformed pre-B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre-B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre-B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc-transformed pre-B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre-B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia.

A genetic, non-transcriptional assay for nuclear receptor ligand binding in yeast.

We describe the development of a genetic, non-transcriptional assay for the detection of ligand binding to nuclear receptors based on the ligand-dependent reconstitution of the defective Ras/cAMP viability pathway of the Saccharomyces cerevisiae strain CDC25-2. We have characterized the assay using the estrogen receptor (ER) alpha as an example and found it to be extremely sensitive, stringent, rapid and selective. We applied this assay to different ligands and ligand-binding domains (LBDs) and analyzed co-stimulation with 17beta-estradiol (E2) and the synthetic ligand 4-hydroxytamoxifen (4-OHT) in vivo. This simple and inexpensive assay may be useful for the study of steroid hormone receptor (SHR) actions at the plasma membrane and for the analysis of ligand binding in vivo. Furthermore, it may allow for the selection of novel ligands and ligand-binding domains and has significant potential for application in compound screening.

A yeast-based growth assay for the analysis of site-specific proteases.

Many cellular and viral processes depend on site-specific proteolysis. Here, a genetic system for the identification of such proteases and inhibitors is described. The system utilizes the temperature- sensitive Saccharomyces cerevisiae CDC25-2 mutant strain and exploits the strict requirement of membrane localization of a constitutively active Ras mutant for the complementation of the yeast growth defect at the non-permissive temperature. Expression of a fusion protein in which a substrate peptide of the TEV protease separates a myristoylation signal from a constitutively active human Ras protein confers temperature insensitivity. Co-expression of the protease results in release of the Ras mutant from the membrane and growth arrest at the non-permissive temperature. This non-transcriptional assay represents a new approach to the in vivo analysis of site-specific proteases and may be a valuable alternative to existing methods. It has significant potential for the selection of inhibitors of cytoplasmic and membrane-associated proteases of biotechnical and clinical relevance.