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1.
Methods Mol Biol ; 2808: 105-120, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743365

RESUMO

Measles is a highly infectious disease that continues to spread mainly in developing countries, often resulting in child mortality. Despite the existence of effective vaccines, no specific antivirals are available as targeted therapy to combat measles virus (MeV). The implementation of genome-wide siRNA screens can provide a powerful platform to discover host factors that mediate MeV infection and replication, which could be essential to develop novel therapeutic strategies against this disease. Here, we describe a human genome-wide siRNA screen for MeV.


Assuntos
Vírus do Sarampo , RNA Interferente Pequeno , Humanos , RNA Interferente Pequeno/genética , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Interações Hospedeiro-Patógeno/genética , Replicação Viral/genética , Genoma Humano , Interferência de RNA
2.
Emerg Microbes Infect ; 12(2): 2256416, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37672505

RESUMO

The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.


Assuntos
COVID-19 , Coronavirus Humano OC43 , Humanos , Coronavirus Humano OC43/genética , SARS-CoV-2 , Receptores de Hidrocarboneto Arílico/genética , Linhagem Celular
3.
PLoS Pathog ; 18(8): e1010763, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35939522

RESUMO

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.


Assuntos
Infecções por Coronavirus , Coronavirus , Dengue , Metabolismo Energético , Humanos , Lipídeos , Proteínas de Membrana/genética , Replicação Viral
4.
Curr Opin Virol ; 50: 159-170, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34488003

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent that causes Coronavirus Disease 2019 (COVID-19) pandemic, is a newly emerging respiratory RNA virus with exceptional transmissibility and pathogenicity. Numerous COVID-19 related studies have been fast-tracked, with the ultimate goal to end the pandemic. Here we review the major stages of SARS-CoV-2 infection cycle in cells, with specific emphasis on essential host factors. Insights into the cell biology of SARS-CoV-2 infection have accelerated the development of host-directed therapeutics, as shown by dozens of clinical trials evaluating COVID-19 treatments using host-targeting compounds.


Assuntos
COVID-19/etiologia , SARS-CoV-2/fisiologia , Catepsina L/fisiologia , Humanos , RNA Viral/biossíntese , SARS-CoV-2/genética , Montagem de Vírus , Internalização do Vírus , Tratamento Farmacológico da COVID-19
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