ABSTRACT
Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS-CoV-2 circRNAs from RNA samples extracted from SARS-CoV-2-infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus-derived circRNAs had a spliceosome-independent origin. We further showed that back-splice junctions (BSJs) captured by inverse reverse-transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ-spanning probe targeting N gene, we identified three RNase R-resistant bands that represent SARS-CoV-2 circRNAs that are detected cytoplasmic by single-molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back-splice and forward-splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.
Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Humans , In Situ Hybridization, Fluorescence , Middle East Respiratory Syndrome Coronavirus/genetics , RNA, Circular/genetics , SARS-CoV-2/genetics , Spliceosomes/geneticsABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the global pandemic of the coronavirus disease-2019 (COVID-19). To date, there are still no effective approaches for the prevention and treatment of COVID-19. OBJECTIVE: The present study aims to explore the possible mechanisms of SARS-CoV-2 infection in human lung cells. METHODS: Data interpretation was conducted by recruiting bioinformatics analysis, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways analysis using downloaded data from the NCBI Gene Expression Omnibus database. RESULTS: The present study demonstrated that SARS-CoV-2 infection induces the upregulation of 14 interferon-stimulated genes, indicative of immune, and interferon responses to the virus. Notably, genes for pyrimidine metabolism and steroid hormone biosynthesis are selectively enriched in human lung cells after SARS-CoV-2 infection, suggesting that altered pyrimidine metabolism and steroid biosynthesis are remarkable, and perhaps druggable features after SARS-CoV-2 infection. Besides, there is a strong positive correlation between viral ORF1ab, ORF6, and angiotensin-converting enzyme 2 (ACE2) expression in human lung cells, implying that ACE2 facilitates SARS-CoV-2 infection and replication in host cells probably through the induction of ORF1ab and ORF6.