Biology subjects

Fontaine, K. A.

Publications and source records attributed to Fontaine, K. A..

4 recordsLinked to original sources

bioRxiv2022

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-{kappa}B inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more I{kappa}B expression in infected than bystander cells, but found concurrent nuclear translocation of NF-{kappa}B that I{kappa}B usually prevents. Overexpressing a nondegradable I{kappa}B mutant reduced NF-{kappa}B translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-{kappa}B feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

microbiology↗

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

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

genetics↗

bioRxiv2018

The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly1, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice2-4. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1)5. Endogenous UPF1 interacted with the viral capsid protein in co-immunoprecipitation experiments and capsid expression post-transcriptionally downregulated UPF1, a process that we confirmed occurs during de novo ZIKV infection. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures. We therefore propose that ZIKV, via the capsid protein, has evolved a strategy to dampen antiviral activities of NMD6,7, which subsequently contributes to neuropathology in vivo.

microbiology↗