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SARS-CoV-encoded small RNAs contribute to infection-associated lung pathology

AuthorsMorales, Lucía ; Oliveros, Juan C.; Fernandez-Delgado, Raúl ; tenOever, Benjamin Robert; Enjuanes Sánchez, Luis ; Solá Gurpegui, Isabel
Issue Date11-Jun-2017
CitationXIV Congreso Nacional de Virología (2017)
AbstractSevere acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. In addition to protein components, viral genomes can also encode non-coding RNAs (ncRNAs) differing in size, biogenesis, and function, which are not necessarily comparable to those produced by host cells. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice, which reproduce the pulmonary pathology observed in humans. Bioinformatics analysis of RNAs sequencesrevealed small RNAs generated from the SARS-CoV genome (svRNAs).Three abundant 18-22 ntsvRNAs derived from the nsp3 (svRNA-nsp3.1 and nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV, which represented 18% outof thetotalsmallviral sequences, were selected for further study. The generation of these svRNAs was confirmed by RTqPCRin different cell types infectedeither by WT or E genemutant SARS-CoV.Production of SARS-CoV svRNAs was cell type and host species independent, but it was dependent on the extent of viral replication. Biogenesis of svRNAs was evaluated in cells defective in Drosha and Dicer enzymes, which are responsible for miRNA canonical processing.svRNAgeneration resulted to be independent of these host RNase IIInucleases,suggesting that svRNA synthesis was mainly dependent on alternative mechanisms involving viral proteins or cellular factors induced during infection. The production of svRNAs during cell culture infection had a positive, although modest effect, on SARSCoV growth. svRNAs specifically repressed the expression of reporter mRNA targets with complementary 3’ UTR sequences, suggesting a post-transcriptional silencing effect of similar character to cellular miRNAs. To assess the contributionof svRNAs in pulmonary pathogenesis, svRNA-N antisense oligonucleotides or antagomirs were intranasally inoculated prior to SARS-CoV infection in mice. Inhibition of svRNA-N in vivo significantly reduced inflammatory cell infiltration and edema observed in the lung of SARS-CoV infected mice in the absence of inhibitors. Accordingly, a significant decrease in the expression of pro-inflammatory cytokines CCL2, IL-6 and CXCL10, associated with SARSCoV pathology, was also observed. Viral titers in the lungs were not affected by svRNA-N Inhibition, indicating that the reduced inflammation should not be attributed to decreased viral growth, but to the contribution of svRNAs to the inflammatory lung pathology induced by SARS-CoV. Together, these results indicate that svRNAs generated from SARS-CoV genome contribute to pathogenesis and highlight the potential of svRNA-N antagomirsin antiviral therapy.
DescriptionTrabajo presentado en el XIV Congreso Nacional de Virología, celebrado en Cádiz (España), del 11 al 14 de junio de 2017
Appears in Collections:(VICYT) Colección Especial COVID-19
(CNB) Comunicaciones congresos
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