Sidelobe modeling and mitigation for a three mirror anastigmat cosmic microwave background telescope.

Telescopes measuring cosmic microwave background (CMB) polarization on large angular scales require exquisite control of systematic errors to ensure the fidelity of the cosmological results. In particular, far-sidelobe contamination from wide angle scattering is a potentially prominent source of systematic error for large aperture microwave telescopes. Here we describe and demonstrate a ray-tracing-based modeling technique to predict far sidelobes for a three mirror anastigmat telescope designed to observe the CMB from the South Pole. Those sidelobes are produced by light scattered in the receiver optics subsequently interacting with the walls of the surrounding telescope enclosure. After comparing simulated sidelobe maps and angular power spectra for different enclosure wall treatments, we propose a highly scattering surface that would provide more than an order of magnitude reduction in the degree-scale far-sidelobe contrast compared to a typical reflective surface. We conclude by discussing the fabrication of a prototype scattering wall panel and presenting measurements of its angular scattering profile.

Comparative Analysis of miRNA Expression after Whole-Body Irradiation Across Three Strains of Mice.

Whole- or partial-body exposure to ionizing radiation damages major organ systems, leading to dysfunction on both acute and chronic timescales. Radiation medical countermeasures can mitigate acute damages and may delay chronic effects when delivered within days after exposure. However, in the event of widespread radiation exposure, there will inevitably be scarce resources with limited countermeasures to distribute among the affected population. Radiation biodosimetry is necessary to separate exposed from unexposed victims and determine those who requires the most urgent care. Blood-based, microRNA signatures have great potential for biodosimetry, but the affected population in such a situation will be genetically heterogeneous and have varying miRNA responses to radiation. Thus, there is a need to understand differences in radiation-induced miRNA expression across different genetic backgrounds to develop a robust signature. We used inbred mouse strains C3H/HeJ and BALB/c mice to determine how accurate miRNA in blood would be in developing markers for radiation vs. no radiation, low dose (1 Gy, 2 Gy) vs. high dose (4 Gy, 8 Gy), and high risk (8 Gy) vs. low risk (1 Gy, 2 Gy, 4 Gy). Mice were exposed to whole-body doses of 0 Gy, 1 Gy, 2 Gy, 4 Gy, or 8 Gy of X rays. MiRNA expression changes were identified using NanoString nCounter panels on blood RNA collected 1, 2, 3 or 7 days postirradiation. Overall, C3H/HeJ mice had more differentially expressed miRNAs across all doses and timepoints than BALB/c mice. The highest amount of differential expression occurred at days 2 and 3 postirradiation for both strains. Comparison of C3H/HeJ and BALB/c expression profiles to those previously identified in C57BL/6 mice revealed 12 miRNAs that were commonly expressed across all three strains, only one of which, miR-340-5p, displayed a consistent regulation pattern in all three miRNA data. Notably multiple Let-7 family members predicted high-dose and high-risk radiation exposure (Let-7a, Let-7f, Let-7e, Let-7g, and Let-7d). KEGG pathway analysis demonstrated involvement of these predicted miRNAs in pathways related to: Fatty acid metabolism, Lysine degradation and FoxO signaling. These findings indicate differences in the miRNA response to radiation across various genetic backgrounds, and highlights key similarities, which we exploited to discover miRNAs that predict radiation exposure. Our study demonstrates the need and the utility of including multiple animal strains in developing and validating biodosimetry diagnostic signatures. From this data, we developed highly accurate miRNA signatures capable of predicting exposed and unexposed subjects within a genetically heterogeneous population as quickly as 24 h of exposure to radiation.

Association of Injury Rates Among Players in the National Football League With Playoff Qualification, Travel Distance, Game Timing, and the Addition of Another Game: Data From the 2017 to 2022 Seasons.

Background: Injury incidence is higher in the National Football League (NFL) than in other collision sports. Although previous research has identified that scheduling variations, including overseas games and bye week timing, does not affect concussion risk, data are currently lacking regarding the effects of scheduling variation on season-long musculoskeletal injury incidence. Purpose: To determine whether higher cumulative travel distance, overseas play, an early season bye week, and an expansion of the regular season is associated with higher injury rates in the NFL. Study Design: Descriptive epidemiology study. Methods: All 1275 injuries across 5 NFL seasons from 2017 to 2018 through 2021 to 2022 were reviewed retrospectively. Injury data and travel distances were extracted from publicly available sources, which were cross-referenced for validation. Injury rates were calculated per 1000 athletic-exposures (AEs). Cumulative team travel distances were compared statistically using a linear regression. Single factor analysis of variance was used to compare categorical variables. Results: Travel distance did not significantly predict injury rates (P = .47), and there was no difference in injury rates between teams that played a game overseas versus teams that did not (19.3 injuries per 1000 AEs for both; P = .96). In addition, no difference was found in injury rates (F[109,2100[ = 0.704; P = .73) or players placed on the injured reserve list (F[99,778] = 1.70; P = .077) between various bye weeks (P = .73). Injury rates did not differ between the new 17-game regular season (18.4 per 1000 AEs) versus the previous four 16-game regular seasons (19.7 per 1000 AEs; P = .12). However, teams that did not qualify for the playoffs had a significantly higher injury rate (19.9 per 1000 AEs) as well as players on injured reserve (8.0 per 1000 AEs) than playoff-qualifying teams (18.4 and 6.8 per 1000 AEs, respectively; P < .05 for both). Conclusion: Over 5 NFL seasons, cumulative travel distance, overseas play, bye week timing, and adding 1 regular season game were not associated with increased injury rates in NFL players. However, a lack of regular season success was associated with higher injury rates and more players on injured reserve.

Microarray analysis of hub genes, non-coding RNAs and pathways in lung after whole body irradiation in a mouse model.

PURPOSE: Previous research has highlighted the impact of radiation damage, with cancer patients developing acute disorders including radiation induced pneumonitis or chronic disorders including pulmonary fibrosis months after radiation therapy ends. We sought to discover biomarkers that predict these injuries and develop treatments that mitigate this damage and improve quality of life. MATERIALS AND METHODS: Six- to eight-week-old female C57BL/6 mice received 1, 2, 4, 8, 12 Gy or sham whole body irradiation. Animals were euthanized 48 h post exposure and lungs removed, snap frozen and underwent RNA isolation. Microarray analysis was performed to determine dysregulation of messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA) after radiation injury. RESULTS: We observed sustained dysregulation of specific RNA markers including: mRNAs, lncRNAs, and miRNAs across all doses. We also identified significantly upregulated genes that can indicate high dose exposure, including Cpt1c, Pdk4, Gdf15, and Eda2r, which are markers of senescence and fibrosis. Only three miRNAs were significantly dysregulated across all radiation doses: miRNA-142-3p and miRNA-142-5p were downregulated and miRNA-34a-5p was upregulated. IPA analysis predicted inhibition of several molecular pathways with increasing doses of radiation, including: T cell development, Quantity of leukocytes, Quantity of lymphocytes, and Cell viability. CONCLUSIONS: These RNA biomarkers might be highly relevant in the development of treatments and in predicting normal tissue injury in patients undergoing radiation treatment. We are conducting further experiments in our laboratory, which includes a human lung-on-a-chip model, to develop a decision tree model using RNA biomarkers.

CASC3 Biomolecular Condensates Restrict Turnip Crinkle Virus by Limiting Host Factor Availability.

The exon-junction complex (EJC) plays a role in post-transcriptional gene regulation and exerts antiviral activity towards several positive-strand RNA viruses. However, the spectrum of RNA viruses that are targeted by the EJC or the underlying mechanisms are not well understood. EJC components from Arabidopsis thaliana were screened for antiviral activity towards Turnip crinkle virus (TCV, Tombusviridae). Overexpression of the accessory EJC component CASC3 inhibited TCV accumulation > 10-fold in Nicotiana benthamiana while knock-down of endogenous CASC3 resulted in a > 4-fold increase in TCV accumulation. CASC3 forms cytoplasmic condensates and deletion of the conserved SELOR domain reduced condensate size 7-fold and significantly decreased antiviral activity towards TCV. Mass spectrometry of CASC3 complexes did not identify endogenous stress granule or P-body markers and CASC3 failed to co-localize with an aggresome-specific dye suggesting that CASC3 condensates are distinct from well-established membraneless compartments. Mass spectrometry and bimolecular fluorescence complementation assays revealed that CASC3 sequesters Heat shock protein 70 (Hsp70-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), two host factors with roles in tombusvirus replication. Overexpression of Hsp70-1 or GAPDH reduced the antiviral activity of CASC3 2.1-fold and 2.8-fold, respectively, and suggests that CASC3 inhibits TCV by limiting host factor availability. Unrelated Tobacco mosaic virus (TMV) also depends on Hsp70-1 and CASC3 overexpression restricted TMV accumulation 4-fold and demonstrates that CASC3 antiviral activity is not TCV-specific. Like CASC3, Auxin response factor 19 (ARF19) forms poorly dynamic condensates but ARF19 overexpression failed to inhibit TCV accumulation and suggests that CASC3 has antiviral activities that are not ubiquitous among cytoplasmic condensates.

Microarray analysis identifies coding and non-coding RNA markers of liver injury in whole body irradiated mice.

Radiation injury from medical, accidental, or intentional sources can induce acute and long-term hepatic dysregulation, fibrosis, and cancer. This long-term hepatic dysregulation decreases quality of life and may lead to death. Our goal in this study is to determine acute changes in biological pathways and discover potential RNA biomarkers predictive of radiation injury. We performed whole transcriptome microarray analysis of mouse liver tissue (C57BL/6 J) 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray to identify significant expression changes in mRNAs, lncRNAs, and miRNAs, We also validated changes in specific RNAs through qRT-PCR. We used Ingenuity Pathway Analysis (IPA) to identify pathways associated with gene expression changes. We observed significant dysregulation of multiple mRNAs across all doses. In contrast, miRNA dysregulation was observed upwards of 2 Gray. The most significantly upregulated mRNAs function as tumor suppressors: Cdkn1a, Phlda3, and Eda2r. The most significantly downregulated mRNAs were involved in hemoglobin synthesis, inflammation, and mitochondrial function including multiple members of Hbb and Hba. The most significantly upregulated miRNA included: miR-34a-5p, miR-3102-5p, and miR-3960, while miR-342-3p, miR-142a-3p, and miR-223-3p were most significantly downregulated. IPA predicted activation of cell cycle checkpoint control pathways and inhibition of pathways relevant to inflammation and erythropoietin. Clarifying expression of mRNA, miRNA and lncRNA at a short time point (48 h) offers insight into potential biomarkers, including radiation markers shared across organs and animal models. This information, once validated in human models, can aid in development of bio-dosimetry biomarkers, and furthers our understanding of acute pathway dysregulation.

Decreased Concussion Incidence Following the Implementation of the Targeting Rules: An Updated Epidemiology of National Football League Concussions From 2017 to 2022.

The incidence of concussions in football, and the ensuing media attention, has garnered scientific investigation, prompted technological advances in protective gear, and altered the rules of the game, including the National Football League's (NFL) "Targeting" rule, which began in 2018, but the impact of these changes is unclear. This study aims to describe the epidemiology of concussions that occurred in five NFL seasons from the 2017-2018 season through the 2021-2022 season and characterize positional differences in rate and games missed. There was a significant decrease (p = 0.02) in total concussions between the 2017-2018 season (102 concussions) and the remaining four seasons (average of 73.80 concussions per year), accounting for a 38% decrease. Offensive and defensive units had decreased concussion rates and average games missed per concussion. Defensive backs (10.46 per 1,000 athlete exposures (AEs)) and tight ends (10.69 per 1,000 AEs) had the highest concussion rates, and the defensive line had the highest average games missed per concussion at 3.97. The introduction of the "Targeting" rule and other rule changes in the NFL in 2018 correlated with a decrease in total concussions per year, total games missed due to concussion, and average games missed per concussion. Offense and defense experienced similar reductions in concussion incidence and severity. Overall, the updated epidemiology of NFL concussions suggests that the incidence of concussions has decreased; however, players continue to experience concussions that require them to miss multiple games.

Serum RNA biomarkers for predicting survival in non-human primates following thoracic radiation.

In a mass radiation exposure, the healthcare system may rely on differential expression of miRNA to determine exposure and effectively allocate resources. To this end, miRNome analysis was performed on non-human primate serum after whole thorax photon beam irradiation of 9.8 or 10.7 Gy with dose rate 600 cGy/min. Serum was collected up to 270 days after irradiation and sequenced to determine immediate and delayed effects on miRNA expression. Elastic net based GLM methods were used to develop models that predicted the dose vs. controls at 81% accuracy at Day 15. A three-group model at Day 9 achieved 71% accuracy in determining if an animal would die in less than 90 days, between 90 and 269 days, or survive the length of the study. At Day 21, we achieved 100% accuracy in determining whether an animal would later develop pleural effusion. These results demonstrate the potential ability of miRNAs to determine thorax partial-body irradiation dose and forecast survival or complications early following whole thorax irradiation in large animal models. Future experiments incorporating additional doses and independent animal cohorts are warranted to validate these results. Development of a serum miRNA assay will facilitate the administration of medical countermeasures to increase survival and limit normal tissue damage following a mass exposure.

Phase separation of a plant virus movement protein and cellular factors support virus-host interactions.

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.

Analysis of lncRNA-miRNA-mRNA expression pattern in heart tissue after total body radiation in a mouse model.

BACKGROUND: Radiation therapy is integral to effective thoracic cancer treatments, but its application is limited by sensitivity of critical organs such as the heart. The impacts of acute radiation-induced damage and its chronic effects on normal heart cells are highly relevant in radiotherapy with increasing lifespans of patients. Biomarkers for normal tissue damage after radiation exposure, whether accidental or therapeutic, are being studied as indicators of both acute and delayed effects. Recent research has highlighted the potential importance of RNAs, including messenger RNAs (mRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) as biomarkers to assess radiation damage. Understanding changes in mRNA and non-coding RNA expression will elucidate biological pathway changes after radiation. METHODS: To identify significant expression changes in mRNAs, lncRNAs, and miRNAs, we performed whole transcriptome microarray analysis of mouse heart tissue at 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray (Gy). We also validated changes in specific lncRNAs through RT-qPCR. Ingenuity Pathway Analysis (IPA) was used to identify pathways associated with gene expression changes. RESULTS: We observed sustained increases in lncRNAs and mRNAs, across all doses of radiation. Alas2, Aplnr, and Cxc3r1 were the most significantly downregulated mRNAs across all doses. Among the significantly upregulated mRNAs were cell-cycle arrest biomarkers Gdf15, Cdkn1a, and Ckap2. Additionally, IPA identified significant changes in gene expression relevant to senescence, apoptosis, hemoglobin synthesis, inflammation, and metabolism. LncRNAs Abhd11os, Pvt1, Trp53cor1, and Dino showed increased expression with increasing doses of radiation. We did not observe any miRNAs with sustained up- or downregulation across all doses, but miR-149-3p, miR-6538, miR-8101, miR-7118-5p, miR-211-3p, and miR-3960 were significantly upregulated after 12 Gy. CONCLUSIONS: Radiation-induced RNA expression changes may be predictive of normal tissue toxicities and may indicate targetable pathways for radiation countermeasure development and improved radiotherapy treatment plans.

Whole blood gene expression within days after total-body irradiation predicts long term survival in Gottingen minipigs.

Gottingen minipigs mirror the physiological radiation response observed in humans and hence make an ideal candidate model for studying radiation biodosimetry for both limited-sized and mass casualty incidents. We examined the whole blood gene expression profiles starting one day after total-body irradiation with increasing doses of gamma-rays. The minipigs were monitored for up to 45 days or time to euthanasia necessitated by radiation effects. We successfully identified dose- and time-agnostic (over a 1-7 day period after radiation), survival-predictive gene expression signatures derived using machine-learning algorithms with high sensitivity and specificity. These survival-predictive signatures fare better than an optimally performing dose-differentiating signature or blood cellular profiles. These findings suggest that prediction of survival is a much more useful parameter for making triage, resource-utilization and treatment decisions in a resource-constrained environment compared to predictions of total dose received. It should hopefully be possible to build such classifiers for humans in the future.

Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs.

We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a -1 ribosomal frameshift event, are 2.7-4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3' regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3' cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3' UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.

Long and short non-coding RNA and radiation response: a review.

Once thought of as arising from "junk DNA," noncoding RNAs (ncRNAs) have emerged as key molecules in cellular processes and response to stress. From diseases such as cancer, coronary artery disease, and diabetes to the effects of ionizing radiation (IR), ncRNAs play important roles in disease progression and as biomarkers of damage. Noncoding RNAs regulate cellular processes by competitively binding DNA, mRNA, proteins, and other ncRNAs. Through these interactions, specific ncRNAs can modulate the radiosensitivity of cells and serve as diagnostic and prognostic biomarkers of radiation damage, whether from incidental exposure in radiotherapy or in accidental exposure scenarios. Analysis of RNA expression after radiation exposure has shown alterations not only in mRNAs, but also in ncRNAs (primarily miRNA, circRNA, and lncRNA), implying an important role in cellular stress response. Due to their abundance and stability in serum and other biofluids, ncRNAs also have great potential as minimally invasive biomarkers with advantages over current biodosimetry methods. Several studies have examined changes in ncRNA expression profiles in response to IR and other forms of oxidative stress. Furthermore, some studies have reported modulation of radiosensitivity by altering expression levels of these ncRNAs. This review discusses the roles of ncRNAs in the radiation response and evaluates prior research on ncRNAs as biomarkers of radiation damage. Future directions and applications of ncRNAs in radiation research are introduced, including the potential for a clinical ncRNA assay for assessing radiation damage and for the therapeutic use of RNA interference (RNAi).

Targeting of viral RNAs by Upf1-mediated RNA decay pathways.

Viral RNAs are susceptible to co-translational RNA decay pathways mediated by the RNA helicase Upstream frameshift 1 (Upf1). Upf1 is a key component in nonsense-mediated decay (NMD), Staufen1-mediated mRNA decay (SMD), and structure-mediated RNA decay (SRD) pathways, among others. Diverse families of viruses have features that predispose them to Upf1 targeting, but have evolved means to escape decay through the action of cis-acting or trans-acting viral factors. Studies aimed at understanding how viruses are subjected to and circumvent NMD have increased our understanding of NMD target selection of host mRNAs. This review focuses on the knowledge gained from studying NMD in viral systems as well as related Upf1-dependent pathways and how these pathways restrict virus replication.

Gene Expression Profiles from Heart, Lung and Liver Samples of Total-Body-Irradiated Minipigs: Implications for Predicting Radiation-Induced Tissue Toxicity.

In the event of a major accidental or intentional radiation exposure incident, the affected population could suffer from total- or partial-body exposures to ionizing radiation with acute exposure to organs that would produce life-threatening injury. Therefore, it is necessary to identify markers capable of predicting organ-specific damage so that appropriate directed or encompassing therapies can be applied. In the current work, gene expression changes in response to total-body irradiation (TBI) were identified in heart, lungs and liver tissue of Göttingen minipigs. Animals received 1.7, 1.9, 2.1 or 2.3 Gy TBI and were followed for 45 days. Organ samples were collected at the end of day 45 or sooner if the animal displayed morbidity necessitating euthanasia. Our findings indicate that different organs respond to TBI in a very specific and distinct manner. We also found that the liver was the most affected organ in terms of gene expression changes, and that lipid metabolic pathways were the most deregulated in the liver samples of non-survivors (survival time <45 days). We identified organ-specific gene expression signatures that accurately differentiated non-survivors from survivors and control animals, irrespective of dose and time postirradiation. At what point did these radiation-induced injury markers manifest and how this information could be used for applying intervention therapies are under investigation.

The Multifunctional Long-Distance Movement Protein of Pea Enation Mosaic Virus 2 Protects Viral and Host Transcripts from Nonsense-Mediated Decay.

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant's vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

RNA virus evasion of nonsense-mediated decay.

Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3' untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3' UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3' UTR. Several additional carmovirus 3' UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5' end of their 3' UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3' UTRs.

RNase III nucleases from diverse kingdoms serve as antiviral effectors.

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 system. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of 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 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, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.

To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA invivo Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation.IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.