Summary A successful cellular response to virus infection is essential for evolutionary survival. In plants, arthropods, and nematodes, cellular antiviral defenses rely on RNA interference (RNAi). Interestingly, the mammalian response to virus is predominantly orchestrated through interferon (IFN)-mediated induction of antiviral proteins. Despite the potency of the IFN system, it remains unclear whether mammals also have the capacity to employ antiviral RNAi. Here we investigate this by disabling either IFN, small RNA function or both activities in the context of virus infection. We find that loss of small RNAs in the context of an in vivo RNA virus infection lowers titers due to reduced transcriptional repression of the host antiviral response. In contrast, enabling a virus with the capacity to inhibit the IFN system results in increased titers. Taken together, we conclude that small RNA silencing is not a physiological contributor to the IFN-mediated cellular response to virus infection.

Eukaryotic cells carry two genomes, nuclear (nDNA) and mitochondrial (mtDNA), which are ostensibly decoupled in their replication, segregation and inheritance. It is increasingly appreciated that heteroplasmy, the occurrence of multiple mtDNA haplotypes in a cell, plays an important biological role, but its features are not well understood. Accurately determining the diversity of mtDNA has been difficult, due to the relatively small amount of mtDNA in each cell (<1% of the total DNA), the intercellular variability of mtDNA content and mtDNA pseudogenes (Numts) in nDNA. To understand the nature of heteroplasmy, we developed Mseek, a novel technique to purify and sequence mtDNA. Mseek yields high purity (>90%) mtDNA and its ability to detect rare variants is limited only by sequencing depth, providing unprecedented sensitivity and specificity. Using Mseek, we confirmed the ubiquity of heteroplasmy by analyzing mtDNA from a diverse set of cell lines and human samples. Applying Mseek to colonies derived from single cells, we find heteroplasmy is stably maintained in individual daughter cells over multiple cell divisions. We hypothesized that the stability of heteroplasmy could be facilitated by intercellular exchange of mtDNA. We explicitly demonstrate this exchange by co-culturing cell lines with distinct mtDNA haplotypes. Our results shed new light on the maintenance of heteroplasmy and provide a novel platform to investigate features of heteroplasmy in normal and diseased states.

Significance Virus infections must be combated at a cellular level. The strategies used to inhibit virus differ dramatically when comparing plants and insects to mammals. Here, we identify an evolutionary conserved antiviral response that is independent of these known defenses. We demonstrate that an RNA nuclease called Drosha is repurposed during virus infection to cleave viral RNA and modulate the cellular environment as a means of inhibiting virus replication.

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system1,2. Present day eukaryotes employ at least two main defense strategies that emerged as a result of this viral shift, namely antiviral RNA interference (RNAi) and the interferon (IFN) system2. Here, we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life, also elicit RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment (SELEX) on this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA dependent RNA polymerases (RdRps) of diverse positive stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including: plants, arthropods, invertebrate chordates, and fish. These data implicate RNase III recognition of viral RNA as an antiviral defense that is independent of, and possibly predates, other known eukaryotic antiviral systems.

SUMMARY Responding to an influenza A virus (IAV) infection demands an effective intrinsic cellular defense strategy to slow replication. To identify contributing host factors to this defense, we exploited the host microRNA pathway to perform an in vivo RNAi screen. To this end, IAV, lacking a functional NS1 antagonist, was engineered to encode individual siRNAs against antiviral host genes in an effort to rescue attenuation. This screening platform resulted in the enrichment of strains targeting virus-activated transcription factors, specific antiviral effectors, and intracellular pattern recognition receptors (PRRs). Interestingly, in addition to RIG-I, the PRR for IAV, a virus with the capacity to silence MDA5 also emerged as a dominant strain in wild type, but not in MDA5-deficient mice. Transcriptional profiling of infected knockout cells confirmed RIG-I to be the primary PRR for IAV but implicated MDA5 as a significant contributor to the cellular defense against influenza A virus.