Quantification of influenza virus mini viral RNA dynamics using Cas13.

Autor:	Lamb CH; Department of Molecular Biology, Princeton University, Princeton, NJ 08544., Pitré EM; Department of Molecular Biology, Princeton University, Princeton, NJ 08544.; University of Cambridge, Department of Pathology, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom., Elshina E; Department of Molecular Biology, Princeton University, Princeton, NJ 08544., Rigby CV; Department of Molecular Biology, Princeton University, Princeton, NJ 08544.; University of Cambridge, Department of Pathology, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom.; Public Health England, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom., Bisht K; Department of Molecular Biology, Princeton University, Princeton, NJ 08544., Oade MS; Department of Molecular Biology, Princeton University, Princeton, NJ 08544., Jalal H; Public Health England, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom., Myhrvold C; Department of Molecular Biology, Princeton University, Princeton, NJ 08544.; Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544.; Omenn-Darling Bioengineering Institute, Princeton University, Princeton, NJ 08544.; Department of Chemistry, Princeton University, Princeton, NJ 08544., Te Velthuis AJW; Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 May 02. Date of Electronic Publication: 2024 May 02.
DOI:	10.1101/2023.11.03.565460
Abstrakt:	Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we use an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification dynamics and subcellular distributions. We show that our assay can quantify the copy numbers of specific mvRNA sequences in infected tissue culture cells, ferret upper and lower respiratory tract tissue infected with two different pandemic H1N1 IAV strains, or clinical nasopharyngeal swab extracts of hospitalized patients infected with seasonal H1N1 or H3N2 strains. In addition, we find dynamic differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as among mvRNAs derived from different segments, during IAV infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and suggest that individual aberrant RNAs are not produced stochastically. Competing Interests: Conflict of Interest C.M. is a co-founder and consultant to Carver Biosciences and holds equity in the company. The other authors have no conflicts of interest to declare.
Databáze:	MEDLINE
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