Sustainable agriculture relies on practices and technologies that combine effectiveness with a minimal environmental footprint. RNA interference (RNAi), a eukaryotic process in which transcript expression is reduced in a sequence-specific manner, can be co-opted for the control of plant pests and pathogens in a topical application system. Double-stranded RNA (dsRNA), the key trigger molecule of RNAi, has been shown to provide protection without the need for integration of dsRNA-expressing constructs as transgenes. Consequently, development of RNA-based biopesticides is gaining momentum as a narrow-spectrum alternative to chemical-based control measures, with pests and pathogens targeted with accuracy and specificity. Limitations for a commercially viable product to overcome include stable delivery of the topically applied dsRNA and extension of the duration of protection. In addition to the research focus on delivery of dsRNA, development of regulatory frameworks, risk identification, and establishing avoidance and mitigation strategies is key to widespread deployment of topical RNAi technologies. Once in place, these measures will provide the crop protection industry with the certainty necessary to expend resources on the development of innovative dsRNA-based products. Readily evident risks to human health appear minimal, with multiple barriers to uptake and a long history of consumption of dsRNA from plant material. Unintended impacts to the environment are expected to be most apparent in species closely related to the target. Holistic design practices, which incorporate bioinformatics-based dsRNA selection along with experimental testing, represent important techniques for elimination of adverse impacts.

/pdf/a-perspective-on-rnai-based-biopesticides-3owkdtufyj.pdf

A Perspective on RNAi-Based Biopesticides.

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for genome RNA replication of enteroviruses, small non-enveloped viruses belonging to the family Picornaviridae. Here, we demonstrated that PI4KB is also essential for genome replication of another picornavirus, Aichi virus (AiV), but is recruited to the genome replication sites by a different strategy from that utilized by enteroviruses. AiV non-structural proteins, 2B, 2BC, 2C, 3A, and 3AB, interacted with a Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3). Furthermore, we identified previously unknown interaction between ACBD3 and PI4KB, which provides a novel manner of Golgi recruitment of PI4KB. Knockdown of ACBD3 or PI4KB suppressed AiV RNA replication. The viral proteins, ACBD3, PI4KB, and phophatidylinositol-4-phosphate (PI4P) localized to the viral RNA replication sites. AiV replication and recruitment of PI4KB to the RNA replication sites were not affected by brefeldin A, in contrast to those in enterovirus infection. These results indicate that a viral protein/ACBD3/PI4KB complex is formed to synthesize PI4P at the AiV RNA replication sites and plays an essential role in viral RNA replication.

/pdf/acbd3-mediated-recruitment-of-pi4kb-to-picornavirus-rna-2l4jerw90s.pdf

ACBD3‐mediated recruitment of PI4KB to picornavirus RNA replication sites

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5'- and 3'-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

/pdf/transfection-of-microrna-mimics-should-be-used-with-caution-3ouwij293u.pdf

Transfection of microRNA Mimics Should Be Used with Caution

S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

https://www.pnas.org/content/pnas/115/15/E3388.full.pdf

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation.

IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In nonprofessional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). Although origin, localization, and length are factors in mediating dsRNA recognition and binding by cellular dsRNA-binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. In this study, we show a dsRNA length-dependent activation of IRFs, IFNs, and IFN-stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IFN-β promoter stimulator 1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IFN-β promoter stimulator 1 and IRF3 dependent and independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60–90% inhibition of virus replication was observed following 24-h treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.

Long Double-Stranded RNA Induces an Antiviral Response Independent of IFN Regulatory Factor 3, IFN-β Promoter Stimulator 1, and IFN

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.

/pdf/the-nitric-oxide-pathway-provides-innate-antiviral-2h5inaqz0o.pdf

The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts.

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Long Double-Stranded RNA Induces an Antiviral Response Independent of IFN Regulatory Factor 3, IFN-β Promoter Stimulator 1, and IFN

The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts.