SRA

Viruses constantly evolve and adapt to the antiviral defenses of their hosts. The biology of viral circumvention of these selective pressures can often be attributed to the acquisition of novel antagonistic gene products or by rapid genome change that prevents host recognition. To study viral evasion of RNA interference (RNAi)-based defenses, we established a robust antiviral system in mammalian cells using recombinant Sendai virus designed to be targeted by endogenous host microRNAs (miRNAs) with perfect complementarity. Using this system, we previously demonstrated the intrinsic ability of positive-strand RNA viruses to escape this selective pressure via homologous recombination, which was not observed in negative-strand RNA viruses. Here we show that given extensive time, escape of miRNA-targeted Sendai virus was enabled by host adenosine deaminase acting on RNA 1 (ADAR1). Independent of the viral transcript targeted, ADAR1 editing resulted in disruption of the miRNA-silencing motif, suggesting an intolerance for extensive RNA:RNA interactions necessary for antiviral RNAi. This was further supported in N. benthamiana, where exogenous expression of ADAR1 interfered with endogenous RNAi. Together, these results suggest that ADAR1 diminishes the effectiveness of RNAi and may explain why it is absent in species that utilize this antiviral defense system. Overall design: RNA-sequencing of mammalian cells lines infected with recombinant Sendai viruses that have sequences with perfectly complemenatrity to host miRNAs added into the 3' UTR of the N gene or P gene to produce an artificial miRNA-based antiviral RNAi. Cell lines, which include A549s, STAT1 KO A549s, STAT1/ADAR1 Double KO A549s, MEFs, and MDCKs were infected and RNA was collected at various timepoints post infection to assess host response and changes to viral genome. Samples where differential gene expression in host cells is assessed are in triplicate.