PKR Transduces MDA5-Dependent Signals for Type I IFN Induction.

Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVΔE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluΔNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2α, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVΔE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism.

Evidence for a cytoplasmic microprocessor of pri-miRNAs.

microRNAs (miRNAs) represent a class of noncoding RNAs that fine-tune gene expression through post-transcriptional silencing. While miRNA biogenesis occurs in a stepwise fashion, initiated by the nuclear microprocessor, rare noncanonical miRNAs have also been identified. Here we characterize the molecular components and unique attributes associated with the processing of virus-derived cytoplasmic primary miRNAs (c-pri-miRNAs). RNA in situ hybridization and inhibition of cellular division demonstrated a complete lack of nuclear involvement in c-pri-miRNA cleavage while genetic studies revealed that maturation still relied on the canonical nuclear RNase III enzyme, Drosha. The involvement of Drosha was mediated by a dramatic relocalization to the cytoplasm following virus infection. Deep sequencing analyses revealed that the cytoplasmic localization of Drosha does not impact the endogenous miRNA landscape during infection, despite allowing for robust synthesis of virus-derived miRNAs in the cytoplasm. Taken together, this research describes a unique function for Drosha in the processing of highly structured cytoplasmic RNAs in the context of virus infection.

Replication in cells of hematopoietic origin is necessary for Dengue virus dissemination.

Dengue virus (DENV) is a mosquito-borne pathogen for which no vaccine or specific therapeutic is available. Although it is well established that dendritic cells and macrophages are primary sites of DENV replication, it remains unclear whether non-hematopoietic cellular compartments serve as virus reservoirs. Here, we exploited hematopoietic-specific microRNA-142 (miR-142) to control virus tropism by inserting tandem target sites into the virus to restrict replication exclusively in this cell population. In vivo use of this virus restricted infection of CD11b+, CD11c+, and CD45+ cells, resulting in a loss of virus spread, regardless of the route of administration. Furthermore, sequencing of the targeted virus population that persisted at low levels, demonstrated total excision of the inserted miR-142 target sites. The complete conversion of the virus population under these selective conditions suggests that these immune cells are the predominant sources of virus amplification. Taken together, this work highlights the importance of hematopoietic cells for DENV replication and showcases an invaluable tool for the study of virus pathogenesis.

In vivo delivery of cytoplasmic RNA virus-derived miRNAs.

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery.

Noncanonical cytoplasmic processing of viral microRNAs.

Cellular utilization of RNA interference (RNAi) as a mechanism to combat virus infection is thought to be restricted to plants and invertebrates. In vertebrates, antiviral defenses are largely dependent on interferons (IFNs), with the use of small RNAs restricted to microRNA (miRNA)-mediated targeting of host transcripts. Here we demonstrate that incorporation of a primary miRNA into a cytoplasmic virus results in the formation of a Dicer-dependent, DGCR8-independent, mature miRNA capable of conferring RNAi-like activity. Processing of the viral mirtron-like product (virtron) is indistinguishable from endogenous miRNA maturation and elicits post-transcriptional gene silencing, albeit at a reduced level. Furthermore, virtrons impose Dicer-dependent, microprocessor-independent, and IFN-independent interference on virus replication in a sequence-specific manner. Taken together, these results suggest the existence of a noncanonical, small-RNA-based activity capable of processing cytoplasmic hairpins and perhaps contributing to the cell's antiviral arsenal.

Antiviral response dictated by choreographed cascade of transcription factors.

The dendritic cell (DC) is a master regulator of immune responses. Pathogenic viruses subvert normal immune function in DCs through the expression of immune antagonists. Understanding how these antagonists interact with the host immune system requires knowledge of the underlying genetic regulatory network that operates during an uninhibited antiviral response. To isolate and identify this network, we studied DCs infected with Newcastle disease virus, which is able to stimulate innate immunity and DC maturation through activation of RIG-I signaling, but lacks the ability to evade the human IFN response. To analyze this experimental model, we developed a new approach integrating genome-wide expression kinetics and time-dependent promoter analysis. We found that the genetic program underlying the antiviral cell-state transition during the first 18 h postinfection could be explained by a single convergent regulatory network. Gene expression changes were driven by a stepwise multifactor cascading control mechanism, where the specific transcription factors controlling expression changed over time. Within this network, most individual genes were regulated by multiple factors, indicating robustness against virus-encoded immune evasion genes. In addition to effectively recapitulating current biological knowledge, we predicted, and validated experimentally, antiviral roles for several novel transcription factors. More generally, our results show how a genetic program can be temporally controlled through a single regulatory network to achieve the large-scale genetic reprogramming characteristic of cell-state transitions.

Degradation of host microRNAs by poxvirus poly(A) polymerase reveals terminal RNA methylation as a protective antiviral mechanism.

The life cycle of several viruses involves host or virally encoded small noncoding RNAs, which play important roles in posttranscriptional regulation. Small noncoding RNAs include microRNAs (miRNAs), which modulate the transcriptome, and small interfering RNAs (siRNAs), which are involved in pathogen defense in plants, worms, and insects. We show that insect and mammalian poxviruses induce the degradation of host miRNAs. The virally encoded poly(A) polymerase, which polyadenylates viral transcripts, also mediates 3' polyadenylation of host miRNAs, resulting in their degradation by the host machinery. In contrast, siRNAs, which are protected by 2'O-methylation (2'OMe), were not targeted by poxviruses. These findings suggest that poxviruses may degrade host miRNAs to promote replication and that virus-mediated small RNA degradation likely contributed to 2'OMe evolution.

The IKK Kinases: Operators of Antiviral Signaling.

The ability of a cell to combat an intracellular pathogen requires a mechanism to recognize the threat and elicit a transcriptional response against it. In the context of virus infection, the cell must take measures to inhibit viral replication, meanwhile, convey warning signals to neighboring cells of the imminent threat. This immune response is predominantly mediated by the production of cytokines, notably, interferon beta (IFNß). IFNß signaling results in the transcriptional induction of over one hundred antiviral gene products whose timely expression renders infected cells more capable of inhibiting virus replication, while providing the uninfected cells with the reinforcements to generate a less permissive cellular environment. Induction of IFNß and many aspects of the antiviral response pivot on the function of the IKK and IKK-related kinases. Despite sharing high levels of homology and some degree of functional redundancy, the classic IKK kinases: IKKα and IKKß, and the IKK-related kinases: TBK1 and IKKɛ, perform distinct roles in regulating the host antiviral defense. These kinases serve as molecular operators in their cooperative ability to integrate incoming cellular cues and act on a range of essential antiviral transcription factors to reshape the cellular transcriptome during infection.

MicroRNA-mediated species-specific attenuation of influenza A virus.

Influenza A virus leads to yearly epidemics and sporadic pandemics. Present prophylactic strategies focus on egg-grown, live, attenuated influenza vaccines (LAIVs), in which attenuation is generated by conferring temperature sensitivity onto the virus. Here we describe an alternative approach to attenuating influenza A virus based on microRNA-mediated gene silencing. By incorporating nonavian microRNA response elements (MREs) into the open-reading frame of the viral nucleoprotein, we generate reassortant LAIVs for H1N1 and H5N1 that are attenuated in mice but not in eggs. MRE-based LAIVs show a greater than two-log reduction in mortality compared with control viruses lacking MREs and elicit a diverse antibody response. This approach might be combined with existing LAIVs to increase attenuation and improve vaccine safety.