Influenza A virus-derived siRNAs increase in the absence of NS1 yet fail to inhibit virus replication

While the issue of whether RNA interference (RNAi) ever forms part of the antiviral innate immune response in mammalian somatic cells remains controversial, there is considerable evidence demonstrating that few, if any, viral small interfering RNAs (siRNAs) are produced in infected cells. Moreover, total inhibition of RNAi by mutational inactivation of key RNAi factors, such as Dicer or Argonaut 2, has been shown to not enhance virus replication. One potential explanation for this lack of inhibitory effect is that mammalian viruses encode viral suppressors of RNAi (VSRs) that are so effective that viral siRNAs are not produced in infected cells. Indeed, a number of mammalian VSRs have been described of which the most prominent is the influenza A virus (IAV) NS1 protein, which has not only been reported to inhibit RNAi in plants and insects but also to prevent the production of viral siRNAs in IAV-infected human cells. Here, we confirm that an IAV mutant that lacks the viral NS1 protein indeed differs from wild type IAV in that it induces the production of readily detectable levels of Dicer-dependent viral siRNAs in infected human cells. However we also demonstrate that these siRNAs have little if any inhibitory effect on IAV gene expression. 293T or Dicer-knockout cells (NoDice) were infected with Influenza A virus PR8 or NS1-mutant PR8, total small RNA from infected cells deep sequenced to determine the presence of viral-derived siRNAs.