Stem-Loop Recognition by DDX17 Facilitates miRNA Processing and Antiviral Defense

SummaryDEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements

RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus

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

The Mammalian Response to Virus Infection Is Independent of Small RNA Silencing

A successful cellular response to virus infection is essential for evolutionary survival. In plants, arthropods, and nematodes, cellular antiviral defenses rely on 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 investigated this by disabling IFN function, 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, these results indicate that small RNA silencing is not a physiological contributor to the IFN-mediated cellular response to virus infection

Correction to "Evaluation of cloud-resolving model intercomparison simulations using TWP-ICE observations: Precipitation and cloud structure"

International audienceIn the paper “Evaluation of cloud-resolving model intercomparison simulations using TWP-ICE observations: Precipitation and cloud structure” by Adam Varble et al. (Journal of Geophysical Research, 116, D12206, doi:10.1029/2010JD015180, 2011), there are several errors in the wording resulting from our mistaken understanding that the dense ice class in the SAM-B and SAM-S simulations corresponded to graupel. Since the paper was published, we have determined that the dense ice in those simulations was instead treated as hail. Accordingly, in paragraphs 31 and 39, “(hail in SAM)” should be added after “graupel.” In the “SAM-B” and “SAM-S” rows of the “Microphysics” column of Table 1, “(i, w, r, g, s)” should read “(i, w, r, h, s)”; “h, hail” should be added to the list of abbreviations in the footnote. In paragraph 10, “graupel, and snow” should read “hail, and snow.” In paragraph 39 (4th line), figure captions for Figures 9, 12, and 16, and the table caption for Table 6, “graupel” should read “graupel or hail.” In the title for Table 6, “Graupel” should read “Graupel or Hail,” and in the “Density” column of the “SAM/UKMO-2M” row, “400 kg m−3” should read “400 or 900 kg m−3.” In paragraph 44, the first sentence and “That said,” in the second sentence should be deleted

In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus

SummaryResponding 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

Evaluation of cloud‐resolving and limited area model intercomparison simulations using TWP‐ICE observations: 1. Deep convective updraft properties

International audienceTen 3‐D cloud‐resolving model simulations and four 3‐D limited area model simulations of an intense mesoscale convective system observed on 23–24 January 2006 during the Tropical Warm Pool‐International Cloud Experiment (TWP‐ICE) are compared with each other and with observed radar reflectivity fields and dual‐Doppler retrievals of vertical wind speeds in an attempt to explain published results showing a high bias in simulated convective radar reflectivity aloft. This high‐bias results from ice water content being large, which is a product of large, strong convective updrafts, although hydrometeor size distribution assumptions modulate the size of this bias. Making snow mass more realistically proportional to D 2 rather than D 3 eliminates unrealistically large snow reflectivities over 40 dBZ in some simulations. Graupel, unlike snow, produces high biased reflectivity in all simulations, which is partly a result of parameterized microphysics but also partly a result of overly intense simulated updrafts. Peak vertical velocities in deep convective updrafts are greater than dual‐Doppler‐retrieved values, especially in the upper troposphere. Freezing of liquid condensate, often rain, lofted above the freezing level in simulated updraft cores greatly contributes to these excessive upper tropospheric vertical velocities. The strongest simulated updraft cores are nearly undiluted, with some of the strongest showing supercell characteristics during the multicellular (presquall) stage of the event. Decreasing horizontal grid spacing from 900 to 100 m slightly weakens deep updraft vertical velocity and moderately decreases the amount of condensate aloft but not enough to match observational retrievals. Therefore, overly intense simulated updrafts may additionally be a product of unrealistic interactions between convective dynamics, parameterized microphysics, and large‐scale model forcing that promote different convective strengths than observed

An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication

SummaryRNA interference (RNAi) has been extensively used to identify host factors affecting virus infection but requires exogenous delivery of short interfering RNAs (siRNAs), thus limiting the technique to nonphysiological infection models and a single defined cell type. We report an alternative screening approach using siRNA delivery via infection with a replication-competent RNA virus. In this system, natural selection, defined by siRNA production, permits the identification of host restriction factors through virus enrichment during a physiological infection. We validate this approach with a large-scale siRNA screen in the context of an in vivo alphavirus infection. Monitoring virus evolution across four independent screens identified two categories of enriched siRNAs: specific effectors of the direct antiviral arsenal and host factors that indirectly dampened the overall antiviral response. These results suggest that pathogenicity may be defined by the ability of the virus to antagonize broad cellular responses and specific antiviral factors