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1.
PLoS Pathog ; 18(8): e1010763, 2022 Aug 08.
Article in English | MEDLINE | ID: covidwho-1987166

ABSTRACT

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.

2.
Nat Genet ; 54(8): 1078-1089, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1960394

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, 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. We uncovered proviral and antiviral factors across highly interconnected host pathways, including 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 that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.


Subject(s)
COVID-19 , Animals , COVID-19/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Epigenesis, Genetic , Humans , Mice , Mucins/genetics , SARS-CoV-2
3.
Cell ; 184(9): 2394-2411.e16, 2021 04 29.
Article in English | MEDLINE | ID: covidwho-1126769

ABSTRACT

SARS-CoV-2 is the cause of a pandemic with growing global mortality. 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 ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.


Subject(s)
Host-Pathogen Interactions , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , COVID-19/virology , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Chlorocebus aethiops , Female , Genome, Viral , Humans , Lung/virology , Male , Mass Spectrometry , Mitochondria/metabolism , Mitochondria/ultrastructure , Proteome/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/ultrastructure , Vero Cells
4.
Cell ; 184(1): 106-119.e14, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1064913

ABSTRACT

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.


Subject(s)
COVID-19/genetics , Coronavirus Infections/genetics , Coronavirus/physiology , Genome-Wide Association Study , Host-Pathogen Interactions , SARS-CoV-2/physiology , A549 Cells , Animals , Biosynthetic Pathways/drug effects , COVID-19/virology , Cell Line , Chlorocebus aethiops , Cholesterol/biosynthesis , Cholesterol/metabolism , Cluster Analysis , Clustered Regularly Interspaced Short Palindromic Repeats , Common Cold/genetics , Common Cold/virology , Coronavirus/classification , Coronavirus Infections/virology , Gene Knockout Techniques , Host-Pathogen Interactions/drug effects , Humans , Mice , Phosphatidylinositols/biosynthesis , Vero Cells , Virus Internalization/drug effects , Virus Replication
5.
Nature ; 588(7837): 223-224, 2020 12.
Article in English | MEDLINE | ID: covidwho-1019756
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