Biology subjects

Carette, J.

Publications and source records attributed to Carette, J..

5 recordsLinked to original sources

bioRxiv2025

Human assembloid model of emergent neurotropic enteroviruses

Enteroviruses (EVs) are the leading cause of viral meningitis in children. Recent outbreaks of non-polio EVs, most notably EV-A71 and EV-D68, have been associated with a polio-like paralysis known as acute flaccid myelitis (AFM). The lack of relevant models that mimic the cellular and functional responses of these human-restricted pathogens has hampered the development of effective treatments. We have previously engineered human stem cell-derived assembloids that recapitulate the neuromuscular connections underlying muscle contractions by integrating human spinal cord/hindbrain organoids (hSpO) and human skeletal muscle. Here, we used organoids and assembloids to investigate polio and non-polio EV pathogenesis. Infection of assembloids with poliovirus (PV), EV-D68 and EV-A71 resulted in loss of muscle contraction for all three viruses, which could be prevented by treatment with an antiviral agent. Yet, despite the convergence on neuronal dysfunction, the cellular targets by which each virus acted differed. More specifically, single cell transcriptomic profiling uncovered divergent cell tropisms between the EVs, and live imaging experiments revealed different modes and kinetics of cell damage. Altogether, we describe a multi-cellular model that captures viral pathogenesis in a human and circuit-relevant context.

neuroscience↗

bioRxiv2022

Structure-function analysis of enterovirus protease 2A in complex with its essential host factor SETD3

Enteroviruses cause a number of medically relevant and widespread human diseases with no approved antiviral therapies currently available. Host-directed therapies present an enticing option for this diverse genus of viruses. We have previously identified the actin histidine methyltransferase SETD3 as a critical host factor physically interacting with the viral protease 2A. Here, we report the 3.5 [A] cryo-EM structure of SETD3 interacting with coxsackievirus B3 2A at two distinct interfaces, including the substrate-binding surface within the SET domain. Structure-function analysis revealed that mutations of key residues in the SET domain resulted in severely reduced binding to 2A and complete protection from enteroviral infection. Our findings provide insight into the molecular basis of the SETD3-2A interaction and a framework for the rational design of host-directed therapeutics against enteroviruses.

biophysics↗

bioRxiv2021

CRISPRa screening with real world evidence identifies potassium channels as neuronal entry factors and druggable targets for SARS-CoV-2

Although vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been successful, there are no good treatments for those who are actively infected. While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from sensory organs and the brain are also susceptible to infection. While many patients suffer from diverse neurological symptoms, the viruss neuronal entry remains mysterious. To discover host factors involved in SARS-CoV-2 viral entry, we performed CRISPR activation (CRISPRa) screens targeting all 6000+ human membrane proteins in cells with and without overexpression of ACE2 using Spike-pseudotyped lentiviruses. This unbiased gain-of-function screening identified both novel and previously validated host factors. Notably, newly found host factors have high expression in neuronal and immune cells, including potassium channel KCNA6, protease LGMN, and MHC-II component HLA-DPB1. We validated these factors using replication-competent SARS-CoV-2 infection assays. Notably, the overexpression of KCNA6 led to a marked increase in infection even in cells with undetectable levels of ACE2 expression. Analysis of human olfactory epithelium scRNA-seq data revealed that OLIG2+/TUJ1+ cells--previously identified as sites of infection in COVID-19 autopsy studies-- have high KCNA6 expression and minimal levels of ACE2. The presence of KCNA6 may thus explain sensory/neuronal aspects of COVID-19 symptoms. Further, we demonstrate that FDA-approved compound dalfampridine, an inhibitor of KCNA-family potassium channels, suppresses viral entry in a dosage-dependent manner. Finally, we identified common prescription drugs likely to modulate the top identified host factors, and performed a retrospective analysis of insurance claims of ~8 million patients. This large cohort study revealed a statistically significant association between top drug classes, particularly those targeting potassium channels, and COVID-19 severity. Taken together, the potassium channel KCNA6 facilitates neuronal entry of SARS-CoV-2 and is a promising target for drug repurposing and development.

genomics↗

bioRxiv2018

Impact of a Patient-Derived Hepatitis C Viral RNA Genome with a Mutated MicroRNA Binding Site

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5 end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester mir-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5 genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5 end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.\n\nAuthor SummaryWith the advent of potent direct-acting antivirals (DAA), hepatitis C virus (HCV) can now be eliminated from the majority of patients, using multidrug therapy with DAAs. However, such DAAs are not available for the treatment of most RNA virus infections. The main problem is the high error rate by which RNA-dependent RNA polymerases copy viral RNA genomes, allowing the selection of mutations that are resistant to DAAs. Thus, targeting host-encoded functions that are essential for growth of the virus but not for the host cell offer promising, novel approaches. HCV needs host-encoded microRNA miR-122 for its viral RNA replication in the liver, and depletion of miR-122 in HCV patients results in loss of viral RNA. This study shows that a single-nucleotide mutation in HCV allows viral RNA amplification when miR-122 abundances are low, concomitant with changes in its tropism.

microbiology↗

bioRxiv2018

ATRAID, a genetic factor that regulates the clinical action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFFs) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase (FDPS), resulting in defects in protein prenylation. Yet it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID. Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Lastly, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare non-synonymous coding variants in patients with ONJ or AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people. One Sentence SummaryATRAID is essential for responses to the commonly prescribed osteoporosis drugs nitrogen-containing bisphosphonates. OverlineBONE

genomics↗