ABSTRACT
Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has led to a global coronavirus disease 2019 (COVID-19) pandemic. Currently, incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs. Results In this study, we found that SARS-CoV-2 hijacks the host protein EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) to promote the activity of its helicase NSP13 to facilitate viral propagation. NSP13 is highly conserved among coronaviruses and is crucial for virus replication, providing chemical energy to unwind viral RNA replication intermediates. Treatment with different SARS-CoV-2 NSP13 inhibitors in multiple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection. Using affinity-purification mass spectrometry, the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13. Furthermore, silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels. Mechanistically, EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability. Conclusion In conclusion, our results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.
ABSTRACT
Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.
Subject(s)
Aminohydrolases/genetics , COVID-19/genetics , Formate-Tetrahydrofolate Ligase/genetics , Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics , Multienzyme Complexes/genetics , Pandemics , Aminohydrolases/antagonists & inhibitors , Animals , Antiviral Agents/therapeutic use , COVID-19/virology , Cell Line , Chiroptera/genetics , Chiroptera/virology , Formate-Tetrahydrofolate Ligase/antagonists & inhibitors , Humans , Methylenetetrahydrofolate Dehydrogenase (NADP)/antagonists & inhibitors , Minor Histocompatibility Antigens , Multienzyme Complexes/antagonists & inhibitors , RNA Viruses/genetics , SARS-CoV-2/pathogenicity , Virus Replication/genetics , COVID-19 Drug TreatmentABSTRACT
New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies' composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual's respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies' profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak.