Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation.

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

CRK and NCK adaptors may functionally overlap in zebrafish neurodevelopment, as indicated by common binding partners and overlapping expression patterns.

CRK adaptor proteins are important for signal transduction mechanisms driving cell proliferation and positioning during vertebrate central nervous system development. Zebrafish lacking both CRK family members exhibit small, disorganized retinas with 50% penetrance. The goal of this study was to determine whether another adaptor protein might functionally compensate for the loss of CRK adaptors. Expression patterns in developing zebrafish, and bioinformatic analyses of the motifs recognized by their SH2 and SH3 domains, suggest NCK adaptors are well-positioned to compensate for loss of CRK adaptors. In support of this hypothesis, proteomic analyses found CRK and NCK adaptors share overlapping interacting partners including known regulators of cell adhesion and migration, suggesting their functional intersection in neurodevelopment.

Long Noncoding RNA U90926 Is Induced in Activated Macrophages, Is Protective in Endotoxic Shock, and Encodes a Novel Secreted Protein.

Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells.

Assessing risk of vector transmission of Chagas disease through blood source analysis using LC-MS/MS for hemoglobin sequence identification.

Chagas disease is mainly transmitted by triatomine insect vectors that feed on vertebrate blood. The disease has complex domiciliary infestation patterns and parasite transmission dynamics, influenced by biological, ecological, and socioeconomic factors. In this context, feeding patterns have been used to understand vector movement and transmission risk. Recently, a new technique using Liquid chromatography tandem mass spectrometry (LC-MS/MS) targeting hemoglobin peptides has showed excellent results for understanding triatomines' feeding patterns. The aim of this study was to further develop the automated computational analysis pipeline for peptide sequence taxonomic identification, enhancing the ability to analyze large datasets data. We then used the enhanced pipeline to evaluate the feeding patterns of Triatoma dimidiata, along with domiciliary infestation risk variables, such as unkempt piles of firewood or construction material, cracks in bajareque and adobe walls and intradomiciliary animals. Our new python scripts were able to detect blood meal sources in 100% of the bugs analyzed and identified nine different species of blood meal sources. Human, chicken, and dog were the main blood sources found in 78.7%, 50.4% and 44.8% of the bugs, respectively. In addition, 14% of the bugs feeding on chicken and 15% of those feeding on dogs were captured in houses with no evidence of those animals being present. This suggests a high mobility among ecotopes and houses. Two of the three main blood sources, dog and chicken, were significantly (p < 0.05) affected by domiciliary infestation risk variables, including cracks in walls, construction material and birds sleeping in the intradomicile. This suggests that these variables are important for maintaining reproducing Triatoma dimidiata populations and that it is critical to mitigate these variables in all the houses of a village for effective control of these mobile vectors.

Crk adaptor proteins are necessary for the development of the zebrafish retina.

BACKGROUND: The development of the central nervous system (CNS) requires critical cell signaling molecules to coordinate cell proliferation and migration in order to structure the adult tissue. Chicken tumor virus #10 Regulator of Kinase (CRK) and CRK-like (CRKL) are adaptor proteins with pre-metazoan ancestry and are known to be required for patterning laminated structures downstream of Reelin (RELN), such as the cerebral cortex, cerebellum, and hippocampus. CRK and CRKL also play crucial roles in a variety of other growth factor and extracellular matrix signaling cascades. The neuronal retina is another highly laminated structure within the CNS that is dependent on migration for proper development, but the cell signaling mechanisms behind neuronal positioning in the retina are only partly understood. RESULTS: We find that crk and crkl have largely overlapping expression within the developing zebrafish nervous system. We find that their disruption results in smaller eye size and loss of retinal lamination. CONCLUSIONS: Our data indicate that Crk adaptors are critical for proper development of the zebrafish neural retina in a crk/crkl dose-dependent manner.

Clockwise and counterclockwise hysteresis characterize state changes in the same aquatic ecosystem.

Incremental increases in a driver variable, such as nutrients or detritus, can trigger abrupt shifts in aquatic ecosystems that may exhibit hysteretic dynamics and a slow return to the initial state. A model system for understanding these dynamics is the microbial assemblage that inhabits the cup-shaped leaves of the pitcher plant Sarracenia purpurea. With enrichment of organic matter, this system flips within three days from an oxygen-rich state to an oxygen-poor state. In a replicated greenhouse experiment, we enriched pitcher-plant leaves at different rates with bovine serum albumin (BSA), a molecular substitute for detritus. Changes in dissolved oxygen (DO) and undigested BSA concentration were monitored during enrichment and recovery phases. With increasing enrichment rates, the dynamics ranged from clockwise hysteresis (low), to environmental tracking (medium), to novel counter-clockwise hysteresis (high). These experiments demonstrate that detrital enrichment rate can modulate a diversity of hysteretic responses within a single aquatic ecosystem, and suggest different management strategies may be needed to mitigate the effects of high vs. low rates of detrital enrichment.

FYN and ABL Regulate the Interaction Networks of the DCBLD Receptor Family.

The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.

Investigating the impact of the phosphorylation status of tyrosine residues within the TACC domain of TACC3 on microtubule behavior during axon growth and guidance.

Axon guidance is a critical process in forming the connections between a neuron and its target. The growth cone steers the growing axon toward the appropriate direction by integrating extracellular guidance cues and initiating intracellular signal transduction pathways downstream of these cues. The growth cone generates these responses by remodeling its cytoskeletal components. Regulation of microtubule dynamics within the growth cone is important for making guidance decisions. TACC3, as a microtubule plus-end binding (EB) protein, modulates microtubule dynamics during axon outgrowth and guidance. We have previously shown that Xenopus laevis embryos depleted of TACC3 displayed spinal cord axon guidance defects, while TACC3-overexpressing spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Tyrosine kinases play an important role in relaying guidance signals to downstream targets during pathfinding events via inducing tyrosine phosphorylation. Here, in order to investigate the mechanism behind TACC3-mediated axon guidance, we examined whether tyrosine residues that are present in TACC3 have any role in regulating TACC3's interaction with microtubules or during axon outgrowth and guidance behaviors. We find that the phosphorylatable tyrosines within the TACC domain are important for the microtubule plus-end tracking behavior of TACC3. Moreover, TACC domain phosphorylation impacts axon outgrowth dynamics such as growth length and growth persistency. Together, our results suggest that tyrosine phosphorylation of TACC3 affects TACC3's microtubule plus-end tracking behavior as well as its ability to mediate axon growth dynamics in cultured embryonic neural tube explants.

Defining the TLT-1 interactome from resting and activated human platelets.

The triggering receptor expressed on myeloid cells (TREM) protein family forms a class of type I transmembrane proteins expressed in immune cells that play important roles in innate and adaptive immune responses. The TREM family member TREM-like transcript 1 (TLT-1, also TREML1) is expressed in megakaryocytes and packaged into platelet granules. TLT-1 binds fibrinogen and plays a role in bleeding initiated by inflammatory insults. Here, we describe a proteomics screen that maps the TLT-1 interactome in resting and activated human platelets. Several identified TLT-1 interactors are involved in cell adhesion and migration, as well as platelet activation. Select interactors, including ß3-integrin, RACK1, GRB2, and Rabs 5A, 7, and 11A, were additionally characterized in co-immunoprecipitation/immunoblotting experiments. Finally, several phosphorylation sites were found on immunoprecipitated TLT-1, including Thr280, a novel, regulated site on a conserved residue near the TLT-1 ITIM regulatory sequence. SIGNIFICANCE: Platelet function relies on the secretion of active molecules from intracellular vesicles, or granules, which contain soluble and membrane-bound proteins that are essential for platelet aggregation, coagulation reactions, and pathogen defense mechanisms. TLT-1 is sequestered in α-granules and transported to the plasma membrane, where it plays a unique role in hemostasis after inflammatory insults. Despite the known importance of TLT-1 in platelet biology, our knowledge of TLT-1 mechanistic signaling is limited. This study defines the TLT-1 interactome in resting and active human platelets, identifying several novel TLT-1 interactors, as well as TLT-1 phosphorylation sites, all with likely signaling implications in platelet aggregation dynamics.

Dimerization of small integral membrane protein 1 promotes cell surface presentation of the Vel blood group epitope.

The Vel blood group antigen is carried on the short extracellular segment of the 78-amino-acid-long, type II transmembrane protein SMIM1 of unknown function. Here, using biochemical analysis and flow cytometry of cells expressing wild-type and mutant alleles of SMIM1, we demonstrate that dimerization of SMIM1 promotes cell surface display of the Vel epitope. We show that SMIM1 dimerization is mediated both by an extracellular Cys77-dependent, homomeric disulfide linkage and via a GxxxG helix-helix interaction motif in the transmembrane domain. These results provide important context for the observed variability in reactivity patterns of clinically important anti-Vel identified in patient sera.

NEDD4 family ubiquitin ligases associate with LCMV Z's PPXY domain and are required for virus budding, but not via direct ubiquitination of Z.

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1ß. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

Protein mass spectrometry detects multiple bloodmeals for enhanced Chagas disease vector ecology.

Chagas disease, a neglected tropical disease endemic in Latin America, is caused by the protozoan parasite Trypanosoma cruzi and is responsible for significant health impacts, especially in rural communities. The parasite is transmitted by insect vectors in the Triatominae subfamily and due to lack of vaccines and limited treatment options, vector control is the main way of controlling the disease. Knowing what vectors are feeding on directly enhances our understanding of the ecology and biology of the different vector species and can potentially aid in engaging communities in active disease control, a concept known as Ecohealth management. We evaluated bloodmeals in rural community, house-caught insect vectors previously evaluated for bloodmeals via DNA analysis as part of a larger collaborative project from three countries in Central America, including Guatemala. In addition to identifying bloodmeals in 100% of all samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) (n = 50), strikingly for 53% of these samples there was no evidence of a recent bloodmeal by DNA-PCR. As individual vectors often feed on multiple sources, we developed an enhanced detection pipeline, and showed the ability to quantify a bloodmeal using stable-isotope-containing synthetic references peptides, a first step in further exploration of species-specific bloodmeal composition. Furthermore, we show that a lower resolution mass spectrometer is sufficient to correctly identify taxa from bloodmeals, an important and strong attribute of our LC-MS/MS-based method, opening the door to using proteomics in countries where Chagas disease is endemic.

PKC induces release of a functional ectodomain of the guidance cue semaphorin6A.

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)-induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact-mediated functions, Sema6A may have regulated, long-range, forward-signaling capacity.

The DCBLD receptor family: emerging signaling roles in development, homeostasis and disease.

The discoidin, CUB, and LCCL domain-containing (DCBLD) receptor family are composed of the type-I transmembrane proteins DCBLD1 and DCBLD2 (also ESDN and CLCP1). These proteins are highly conserved across vertebrates and possess similar domain structure to that of neuropilins, which act as critical co-receptors in developmental processes. Although DCBLD1 remains largely uncharacterized, the functional and mechanistic roles of DCBLD2 are emerging. This review provides a comprehensive discussion of this presumed receptor family, ranging from structural and signaling aspects to their associations with cancer, physiology, and development.

Host-Driven Phosphorylation Appears to Regulate the Budding Activity of the Lassa Virus Matrix Protein.

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the virus life cycle, including coordinating viral assembly, driving the release of new virus particles, regulating viral polymerase activity, and antagonizing the host antiviral response. There is limited knowledge regarding how the various functions of Z are regulated. To investigate possible means of regulation, mass spectrometry was used to identify potential sites of phosphorylation in the LASV Z protein. This analysis revealed that two serines (S18, S98) and one tyrosine (Y97) are phosphorylated in the flexible N- and C-terminal regions of the protein. Notably, two of these sites, Y97 and S98, are located in (Y97) or directly adjacent to (S98) the PPXY late domain, an important motif for virus release. Studies with non-phosphorylatable and phosphomimetic Z proteins revealed that these sites are important regulators of the release of LASV particles and that host-driven, reversible phosphorylation may play an important role in the regulation of LASV Z protein function.

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A.

The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.