Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2.

COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.

ILF3 Is a Negative Transcriptional Regulator of Innate Immune Responses and Myeloid Dendritic Cell Maturation.

APCs such as myeloid dendritic cells (DCs) are key sentinels of the innate immune system. In response to pathogen recognition and innate immune stimulation, DCs transition from an immature to a mature state that is characterized by widespread changes in host gene expression, which include the upregulation of cytokines, chemokines, and costimulatory factors to protect against infection. Several transcription factors are known to drive these gene expression changes, but the mechanisms that negatively regulate DC maturation are less well understood. In this study, we identify the transcription factor IL enhancer binding factor 3 (ILF3) as a negative regulator of innate immune responses and DC maturation. Depletion of ILF3 in primary human monocyte-derived DCs led to increased expression of maturation markers and potentiated innate responses during stimulation with viral mimetics or classic innate agonists. Conversely, overexpression of short or long ILF3 isoforms (NF90 and NF110) suppressed DC maturation and innate immune responses. Through mutagenesis experiments, we found that a nuclear localization sequence in ILF3, and not its dual dsRNA-binding domains, was required for this function. Mutation of the domain associated with zinc finger motif of ILF3's NF110 isoform blocked its ability to suppress DC maturation. Moreover, RNA-sequencing analysis indicated that ILF3 regulates genes associated with cholesterol homeostasis in addition to genes associated with DC maturation. Together, our data establish ILF3 as a transcriptional regulator that restrains DC maturation and limits innate immune responses through a mechanism that may intersect with lipid metabolism.

Molecular mechanisms of organelle inheritance: lessons from peroxisomes in yeast.

Preserving a functional set of cytoplasmic organelles in a eukaryotic cell requires a process of accurate organelle inheritance at cell division. Studies of peroxisome inheritance in yeast have revealed that polarized transport of a subset of peroxisomes to the emergent daughter cell is balanced by retention mechanisms operating in both mother cell and bud to achieve an equitable distribution of peroxisomes between them. It is becoming apparent that some common mechanistic principles apply to the inheritance of all organelles, but at the same time, inheritance factors specific for each organelle type allow the cell to differentially and specifically control the inheritance of its different organelle populations.

Characterization of Peroxisomal Regulation Networks.

Peroxisome proliferation involves signal recognition and computation by molecular networks that direct molecular events of gene expression, metabolism, membrane biogenesis, organelle proliferation, protein import, and organelle inheritance. Peroxisome biogenesis in yeast has served as a model system for exploring the regulatory networks controlling this process. Yeast is an outstanding model system to develop tools and approaches to study molecular networks and cellular responses and because the mechanisms of peroxisome biogenesis and key aspects of the transcriptional regulatory networks are remarkably conserved from yeast to humans. In this chapter, we focus on the complex regulatory networks that respond to environmental cues leading to peroxisome assembly and the molecular events of organelle assembly. Ultimately, understanding the mechanisms of the entire peroxisome biogenesis program holds promise for predictive modeling approaches and for guiding rational intervention strategies that could treat human conditions associated with peroxisome function.

Peroxins Pex30 and Pex29 Dynamically Associate with Reticulons to Regulate Peroxisome Biogenesis from the Endoplasmic Reticulum.

Peroxisome proliferation occurs by at least two routes, division of existing peroxisomes and de novo biogenesis from the endoplasmic reticulum (ER). The proteins and molecular mechanisms governing peroxisome emergence from the ER are poorly characterized. In this study, we report that two integral membrane peroxins (proteins required for peroxisome biogenesis) in Saccharomyces cerevisiae, Pex29 and Pex30, reside in distinct regions of the ER and associate with Rtn1 and Yop1, reticulon family members that contribute to ER morphology, to govern peroxisome emergence from the ER. In vivo and in vitro analyses reveal that peroxisome proliferation is therefore not restricted to the peroxisome but begins at the level of the ER.

An ancestral role in peroxisome assembly is retained by the divisional peroxin Pex11 in the yeast Yarrowia lipolytica.

The peroxin Pex11 has a recognized role in peroxisome division. Pex11p remodels and elongates peroxisomal membranes prior to the recruitment of dynamin-related GTPases that act in membrane scission to divide peroxisomes. We performed a comprehensive comparative genomics survey to understand the significance of the evolution of the Pex11 protein family in yeast and other eukaryotes. Pex11p is highly conserved and ancestral, and has undergone numerous lineage-specific duplications, whereas other Pex11 protein family members are fungal-specific innovations. Functional characterization of the in-silico-predicted Pex11 protein family members of the yeast Yarrowia lipolytica, i.e. Pex11p, Pex11Cp and Pex11/25p, demonstrated that Pex11Cp and Pex11/25p have a role in the regulation of peroxisome size and number characteristic of Pex11 protein family members. Unexpectedly, deletion of PEX11 in Y. lipolytica produces cells that lack morphologically identifiable peroxisomes, mislocalize peroxisomal matrix proteins and preferentially degrade peroxisomal membrane proteins, i.e. they exhibit the classical pex mutant phenotype, which has not been observed previously in cells deleted for the PEX11 gene. Our results are consistent with an unprecedented role for Pex11p in de novo peroxisome assembly.

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Introduction: Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. Methods: To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Results: Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Discussion: Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

Automated, image-based quantification of peroxisome characteristics with perox-per-cell.

perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually-quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. The software is available at https://github.com/AitchisonLab/perox-per-cell.

Emergent complexity in Myosin V-based organelle inheritance.

How is adaptability generated in a system composed of interacting cellular machineries, each with a separate and functionally critical job to perform? The machinery for organelle inheritance is precisely one such system, requiring coordination between robust and ancient cellular modules, including the cell cycle, cytoskeleton, and organelle biogenesis/identity. Budding yeasts have emerged as powerful models to study these processes, which are critical for cellular survival, propagation, and differentiation, as organelles must compete for access to myosin V motors that travel along polarized actin cables to vectorially deliver bound cargo to the bud. Under the direction of the cell cycle, myosin V motors are recruited to organelles by specific interactions between their carboxyl-terminal globular tail domains and organelle-specific receptors. We used comparative genomics, phylogenetics, and secondary structure modeling to characterize the evolutionary history of these organelle-specific receptors. We find that while some receptors are retained widely across the animals and fungi, others are limited primarily to the Saccharomycetaceae family of budding yeast, with the emergent pattern of a conserved biogenic and inheritance factor often paired with an evolutionarily novel inheritance adaptor. We propose an evolutionary model whereby the emergence of myosin V-based organelle inheritance has utilized mechanisms of paralogy, mutation, and the appearance of pliable evolutionarily novel adaptor proteins. Our findings suggest an overarching evolutionary mechanism for how diverse cargoes compete for a single myosin V motor in organelle transport and detail one system's solution to obtaining evolutionary adaptability amongst constrained cellular modules.

Predicting host-based, synthetic lethal antiviral targets from omics data.

Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection. Thus, we hypothesized that genes depleted in CRISPR KO screens of virus-infected cells may be enriched in SL partners of proteins altered by infection. To investigate this, we established a computational pipeline predicting SL drug targets of viral infections. First, we identified SARS-CoV-2-induced changes in gene products via a large compendium of omics data. Second, we identified SL partners for each altered gene product. Last, we screened CRISPR KO data for SL partners required for cell viability in infected cells. Despite differences in virus-induced alterations detected by various omics data, they share many predicted SL targets, with significant enrichment in CRISPR KO-depleted datasets. Comparing data from SARS-CoV-2 and influenza infections, we found possible broad-spectrum, host-based antiviral SL targets. This suggests that CRISPR KO data are replete with common antiviral targets due to their SL relationship with virus-altered states and that such targets can be revealed from analysis of omics datasets and SL predictions.

Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin.

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication.

The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.

Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality.

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.

Low rate of SARS-CoV-2 incident infection identified by weekly screening PCR in a prospective year-long cohort study.

BACKGROUND: Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear. METHODS: To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys. RESULTS: Incident SARS2-CoV-2 infections were identified in 9/553 (1.6%) participants. Comparisons of SARS2-CoV-2(+) to SARS2-CoV-2(-) participants revealed significantly more close contacts outside the household (median: 5 versus 3; p = 0.005). The incidence of infection was higher among unvaccinated/partially vaccinated than among fully vaccinated participants (9/7,679 versus 0/6,845 person-weeks; p = 0.004). At notification of positive test result, eight cases were symptomatic and one pre-symptomatic. CONCLUSIONS: These data suggest that weekly SARS2-CoV2 surveillance by rtPCR did not efficiently detect pre-symptomatic infections in unvaccinated participants.

Peroxisome biogenesis: something old, something new, something borrowed.

Eukaryotic cells are characterized by their varied complement of organelles. One set of membrane-bound, usually spherical compartments are commonly grouped together under the term peroxisomes. Peroxisomes function in regulating the synthesis and availability of many diverse lipids by harnessing the power of oxidative reactions and contribute to a number of metabolic processes essential for cellular differentiation and organismal development.

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2.

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.