An exploration of host factors of mitochondrial mvRNA localization using LbuCas13a-based detection

Abstract

Influenza A Virus (IAV) infection poses an ongoing threat to human health. The IAV genome is contained in its negative-sense RNA, which replicates using its RNA-dependent RNA polymerase. However, during IAV genome replication, shorter aberrant RNA such as mini viral RNA (mvRNA) can be produced. Previous study has found that mvRNAs PA-60 and PA-66 were detected in mitochondrial fractions of infected cells. However, the method of transportation of mvRNA to mitochondria is not yet understood. In order to detect mvRNA, an LbuCas13abased detection system was optimized for mvRNA detection, testing for conditions including Mg2+ source, Mg2+ concentration, pH-stabilizing and crowding agent buffers, and LbuCas13a:crRNA ratios. It was found that only LbuCas13a:crRNA ratio yielded a significant difference in mvRNA detection. This research also sought to determine the method of localization of mvRNA to mitochondria using components of the innate immune signaling pathway. Retinoic acid-inducible gene 1 or mitochondrial antiviral signaling protein were knocked out in cells expressing the synthetic mvRNA NP61. mvRNA detection found no significant difference in mitochondrial detection in the absence of retinoic acid-inducible gene 1 but potentially suggests an increase in mitochondrial detection in the absence of mitochondrial antiviral signaling protein. These results provide insights into optimal conditions of mvRNA detection using LbuCas13a, and a potential relationship between the innate immune signaling pathway’s involvement in mvRNA mitochondrial localization.