Biology subjects

Carette, J. E.

Publications and source records attributed to Carette, J. E..

13 recordsLinked to original sources

bioRxiv2025

Dissecting the host determinants of flavivirus infection using QIC-seq

Flaviviruses are genetically related, yet cause distinct disease patterns ranging from hepatitis and vascular shock syndrome to encephalitis and congenital abnormalities. There is an incomplete understanding of the cellular pathways co-opted by flaviviruses, and differences in host response to infection may underlie the diverse pathologies caused. We present a single-cell approach (Quantification of Infection and CRISPR guide sequencing; QIC-seq) that combines CRISPR/Cas9 knockout with virus-inclusive transcriptomics to systematically compare host factor requirements and host transcriptional response to flaviviral challenge. We show that dengue and yellow fever viruses are strictly dependent on subunits of the oligosaccharyltransferase complex, while the more distantly related West Nile and Langat viruses are dependent on components of the ER-associated degradation machinery. Our data further shows virus-induced upregulation of interferon-stimulated genes, and activation of the unfolded protein response. Together, QIC-seq enables quantitative comparisons of viral host factor utilization, which may inform development of host-directed antiviral therapies.

microbiology↗

bioRxiv2023

The activated plant NRC4 immune receptor forms a hexameric resistosome

Innate immune responses against microbial pathogens in both plants and animals are regulated by intracellular receptors known as Nucleotide-binding Leucine-rich Repeats (NLR) proteins. In plants, these NLRs play a crucial role in recognizing pathogen effectors, thereby initiating the activation of immune defense mechanisms. Notably, certain NLRs serve as "helper" NLR immune receptors (hNLR), working in tandem with "sensor" NLR immune receptors (sNLR) counterparts to orchestrate downstream signaling events to express disease resistance. In this study, we reconstituted and determined the cryo-EM structure of the hNLR required for cell death 4 (NRC4) resistosome. The auto-active NRC4 formed a previously unanticipated hexameric configuration, triggering immune responses associated with Ca2+ influx into the cytosol. Furthermore, we uncovered a dodecameric state of NRC4, where the coil-coil (CC) domain is embedded within the complex, suggesting an inactive state, and expanding our understanding of the regulation of plant immune responses. One Sentence SummaryThe hexameric NRC4 resistosome mediates cell death associated with cytosolic Ca2+ influx.

plant biology↗

bioRxiv2023

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation

The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation. SIGNIFICANCEHerpesviruses are masters of immune evasion. Most often, they hijack host cellular pathways to modulate the antiviral immune response. Varicellovirus UL49.5 orthologs have evolved as inhibitors of the transporter associated with antigen processing (TAP) and, this way, major modulators of the MHC class I-restricted antigen presentation. This study identifies the long-sought molecular mechanism exploited by bovine herpesvirus 1-encoded UL49.5 to trigger proteasomal degradation of TAP. Our findings demonstrate that the viral protein hijacks host cell CRL2-ubiquitin conjugation and ER-associated degradation pathways to promote TAP degradation. These findings advance the understanding of how herpesviruses can manipulate the cellular machinery.

cell biology↗

bioRxiv2021

Loquacious Modulates Flaviviral RNA Replication in Mosquito Cells

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins SEC61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both SEC61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3 end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells. Author SummaryThere is a wealth of information that dictates virus-host interactions in flavivirus-infected mammalian cells, yet there is only sparse information on the mechanisms that modulate viral gene expression in the mosquito host. Using a novel RNA-protein detection assay, the interactions of SEC61A1 and Loqs with the dengue viral genome were found to have proviral functions in infected mosquito cells. In particular, Loqs forms complexes with the positive-strand of the viral RNA and the very 3 end of the negative-strand viral RNA. Further analyses showed that Loqs modulates viral RNA replication of dengue virus and gene amplification of several other flaviviral genomes. These findings argue that Loqs is an essential proviral host factor in mosquitos.

microbiology↗

bioRxiv2021

Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection

SARS-CoV-2 can cause a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of the host factors mediating viral infection or restriction is critical to elucidate SARS-CoV-2 host-pathogen interactions and the progression of COVID-19. To this end, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. These screens uncovered proviral and antiviral host factors across highly interconnected host pathways, including components implicated in clathrin transport, inflammatory signaling, cell cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high-molecular weight glycoproteins, as a prominent viral restriction network. We demonstrate that multiple membrane-anchored mucins are critical inhibitors of SARS-CoV-2 entry and are upregulated in response to viral infection. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and suggests interactions between SARS-CoV-2 and airway mucins of COVID-19 patients as a host defense mechanism.

genetics↗

bioRxiv2020

Zika Virus Infection Prevents Host mRNA Nuclear Export by Disrupting UPF1 Function

Zika virus (ZIKV) is a mosquito-borne RNA virus that can infect fetuses in utero causing characteristic neurodevelopmental disorders including microcephaly. We previously showed that ZIKV infection downregulates expression of up-frameshift protein 1 (UPF1), a helicase/ATPase and central regulator of the nonsense-mediated mRNA decay pathway. Here, we identify a novel function of nuclear UPF1 in mRNA export. Using crosslinking immunoprecipitation of UPF1 followed by sequencing of associated transcripts as well as fluorescence in situ hybridization experiments, we find widespread mRNA accumulation in the nucleus of human neural progenitor cells (NPCs) upon ZIKV infection or UPF1 knockdown. Knockdown of FREM2, a top UPF1 target transcript encoding an extra-cellular matrix protein critical in fetal development, decreased expression of pluripotency markers and increased expressed neuronal differentiation in NPCs, consistent with the model that trapping FREM2 mRNA in the nucleus perturbs proper NPC function. Collectively, our data uncover a new posttranscriptional mechanism by which ZIKV "shuts off" host mRNA export via UPF1. As we find UPF1 linked to many neurodevelopment pathways, we propose that the lack of host mRNA export contributes to the neurodevelopmental defects associated with ZIKV infection.

microbiology↗

bioRxiv2020

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. Highlights{middle dot} ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species {middle dot} Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways {middle dot} Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function {middle dot} Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection

microbiology↗

bioRxiv2020

Sphingolipid Biosynthesis Inhibition As A Host Strategy Against Diverse Pathogens

Chloroquine is an anti-malarial and immunosuppressant drug that has cationic amphipathic chemical properties. We performed genome-wide screens in human cells with chloroquine and several other widely used cationic amphipathic drugs (CADs) including the anti-depressants, sertraline (Zoloft) and fluoxetine (Prozac), the analgesic nortriptyline (Pamelor), the anti-arrhythmic amiodarone (Cordarone), and the anti-hypertensive verapamil (Calan) to characterize their molecular similarities and differences. Despite CADs having different disease indications but consistent with them sharing key chemical properties, we found CADs to have remarkably similar phenotypic profiles compared with non-CADs we and others have previously screened (1-5). The most significant genetic interaction for all CADs was the initiating step in sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT). A comparison of genome-wide screens performed with diverse pathogens from viruses, bacteria, plants, and parasites including Ebola (6), adeno-associated virus AAV2 (7), HIV (8), Rotavirus (9), Influenza A (10), Zika virus (11), Picornavirus (12), Exotoxin A (13), Cholera toxin (14), Type III secretion system and Shiga toxin (15, 16), Ricin toxin (17), and Toxoplasma gondii (18) showed SPT as a top common host factor and 80% overlap overall in top hits specifically with CADs. Potential sphingolipid-mediated mechanisms for the host response- and virulence-modulating effects of CADs involve autophagy and SERPINE1/PAI-1 (plasminogen activator inhibitor-1). Chloroquine has recently shown potential as an anti-viral agent for the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease (19, 20). Our study demonstrates that numerous readily available drugs molecularly function highly similar to chloroquine, which suggests they might be considered for further pre-clinical investigation in the context of SARS-CoV-2. More generally, our work suggests the diverse pathogen mitigating potential of drugs that inhibit host sphingolipid biosynthesis such as CADs. Brief SummaryOur study demonstrates that numerous readily available drugs molecularly function highly similar to chloroquine, which suggests they might be considered for further pre-clinical investigation in the context of SARS-CoV-2.

genomics↗

bioRxiv2020

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase (DHODH) as its target, along with the discovery that knockdown of genes in pyrimidine salvage pathways sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was efficiently converted into its triphosphates in cells. When combined with GSK983, it led to large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral genome infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activities of RdRp inhibitors.

biochemistry↗

bioRxiv2019

A memory of RPS25 loss drives resistance phenotypes

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Many cell types can nonetheless withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. However, the direct and indirect consequences from sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation.

molecular biology↗

bioRxiv2019

Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells and in vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

molecular biology↗

bioRxiv2018

Immuno-proteomic interrogation of dengue infection reveals novel HLA haplotype-specific MHC-I antigens

Broadly effective vaccines against dengue virus (DENV) infection have remained elusive, despite rising infection rates in the developing world. Infection-specific peptide ligands presented on Major Histocompatibility Complexes (MHC) open new avenues for developing T-cell-based interventions. Past efforts towards mapping viral MHC epitopes were based on computational predictions that only partially reflected actual antigen presentation. To empirically identify DENV-specific MHC ligands, we developed an immuno-proteomics approach for interrogating DENV- and self-derived MHC ligands from infected B-lymphocytes. Here, we report four fundamental findings: First, over 700 infection-specific MHC-ligands reflected host cellular responses to DENV that were not apparent from the proteome. Second, we report 121 viral MHC-I ligands (108 novel) which clustered into discrete hotspots across the DENV polyprotein, some of which spanned DENV polyprotein components, described here as MHC ligands for the first time. Third, we found DENV ligands which were distinctly presented by MHC alleles previously associated with either high or low anti-DENV response. Fourth, we demonstrate that while our in vitro assay only overlapped with a small fraction of previously described DENV T-cell epitopes, several novel MHC ligands identified here were recognized by T-cells from DENV-infected patients despite having low binding affinities. Together, these discoveries suggest that virus and host-derived MHC ligands have under-exploited potential for describing the cell biology of DENV infection, and as candidates for designing effective DENV vaccines.

immunology↗

bioRxiv2018

Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens

The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

bioinformatics↗