Impact of the structural integrity of the three-way junction of adenovirus VAI RNA on PKR inhibition

Sherpa Romeo green journal. Open access article. Creative Commons Attribution License applies.Highly structured RNA derived from viral genomes is a key cellular indicator of viral infection. In response, cells produce the interferon inducible RNA-dependent protein kinase (PKR) that, when bound to viral dsRNA, phosphorylates eukaryotic initiation factor 2αand attenuates viral protein translation. Adenovirus can evade this line of defence through transcription of a non-coding RNA, VAI, an inhibitor of PKR. VAI consists of three base-paired regions that meet at a three-way junction; an apical stem responsible for the interaction with PKR, a central stem required for inhibition, and a terminal stem. Recent studies have highlighted the potential importance of the tertiary structure of the three-way junction to PKR inhibition by enabling interaction between regions of the central and terminal stems. To further investigate the role of the three-way junction, we characterized the binding affinity and inhibitory potential of central stem mutants designed to introduce subtle alterations. These results were then correlated with small-angle X-ray scattering solution studies and computational tertiary structural models. Our results demonstrate that while mutations to the central stem have no observable effect on binding affinity to PKR, mutations that appear to disrupt the structure of the three-way junction prevent inhibition of PKR. Therefore, we propose that instead of simply sequestering PKR, a specific structural conformation of the PKR-VAI complex may be required for inhibition

Activation of 2′ 5′-oligoadenylate synthetase by stem loops at the 5′-end of the West Nile virus genome

West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII) whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response

Impact of double-stranded RNA characteristics on the activation of human 2’-5’-oligoadenylate synthetase 2 (OAS2).

Human 2’-5’ oligoadenylate synthetases (OAS) are interferon inducible proteins which, upon activation by double-stranded RNA, polymerize ATP into 2’-5’ linked oligoadenylates. Here, we probe the RNA cofactor specificity of the two smallest isozymes, OAS1 and OAS2. First, we demonstrate the purification of recombinant OAS2 from human cells and quantified enzymatic activity relative to OAS1. We then confirmed that both OAS2 domains, as opposed to only the domain containing the canonical catalytic aspartic acid triad, are required for enzymatic activity. Enzyme kinetics of both OAS1/OAS2 in the presence of RNA binding partners enabled characterization of the maximum reaction velocity and apparent RNA-protein affinity of activating RNAs. While OAS1 can be activated by dsRNA greater than 19bp, OAS2 showed a marked increase in activity with increasing dsRNA length with a minimum requirement of 35bp. Interestingly, activation of OAS2 was also more efficient when the dsRNA contained 3’-overhangs despite no significant impact on binding affinity. Highly structured viral RNAs that are established OAS1 activators were not able to activate OAS2 enzymatic activity based on the lack of extended stretches of dsRNA of greater than 35 bp. Together these results may highlight distinct subsets of biological RNAs to which different human OAS isozymes respond.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

UAVs path planning architecture for effective medical emergency response in future networks

With the advancements of the Unmanned Aerial Vehicles (UAV) technology for use in different environments, it can be easily substituted for traditional transportation in event of emergencies. In the medical domain, UAV can play a vital role in the fast and efficient delivery of first aid and medical supplies. In the current study, safe and smooth UAV navigation from the initial position to the medical emergency location was achieved with optimal path planning through a proposed algorithm. On the notification of patient about his health condition using GSM band, doctor drone was sent from the nearest hospital facility. To avoid traffic congestion the doctor drone provides medical assistance with minimum computational time and transportation cost. The vehicle routing was carried out through proposed algorithms i.e., capacitated Vehicle Routing Problem (CVRP), Particle Swarm Optimization (PSO), Ant Colony Optimization (ACO) and Genetic Algorithm (GA). The comparison between the algorithms was carried out at different vehicle capacities and numbers. The CVRP was found to outperform other algorithms with a runtime of 0.06 sec and cost of 419 at vehicle capacity 10, which is 50% less having the same number of the vehicles but increasing the capacities to 20. The results indicate that the effective path planning method could be applied to provide medical aid in real-time with efficacy

Genetic Variability Studies in Brinjal (Solanum melongena L.) for Quantitative and Qualitative Characters

A study was carried out on brinjal to estimate variability, heritability, and genetic advance in the Department of Vegetable Science, College of Horticulture and Forestry, Pasighat. The high Genotypic coefficient of variation (GCV) and Phenotypic coefficient of variation (PCV) (>20%) values for fruit diameter, fruit length, fruit weight, number of branches per plant, leaf area, number of fruits per plant, number of seeds per fruit, total phenol, anthocyanin, and solasodine content indicated high genetic variability among genotypes, making selection more effective for brinjal improvement. High heritability and high genetic advances were seen in the number of seeds per fruit, fruit yield per ha, leaf area, fruit weight, anthocyanin, and total phenol, which may be owing to additive gene action and selection from these traits being more successful

Evaluation of Morphological Traits in Cowpea [Vigna unguiculata (L.) Walp.] Genotypes Cultivated in North-East India

The present study was conducted during the kharif season 2022 at the Vegetable Research Farm, located within the College of Horticulture and Forestry at Central Agricultural University in Pasighat, East Siang, Arunachal Pradesh, India to investigate “Evaluation of Morphological Traits in Cowpea [Vigna unguiculata (L.) Walp.] Genotypes Cultivated in North-East India”. These morphological traits demonstrated significant variation among the twenty locally accessible genotypes. Qualitative traits such as growth habit, twining habit, foliage colour, leaflet shape, flower colour, pod shape, pod colour, seed shape, seed colour were recorded. Quantitative characters such as days to 50% flowering, plant height (cm), leaf area (cm²), number of clusters per plant, number of pods per cluster, number of pods per plant, pod length (cm), pod width (cm), days to harvest, number of seeds per pod, 100 seed weight (g) and pod yield per plant (g) were studied. The mean performance revealed that CHF CP-12 gave highest pod yield per plant (589.19 g), highest 100 seed weight in CHF CP-12 (24.67 g), longest pod length in CHF CP-5 (32.77 cm), longest pod width in CHF CP-10 (1.07 cm), maximum number of clusters per plant was recorded in CHF CP-12 (14.16), maximum number of pods per plant in CHF CP-17 (11.80) and maximum leaf area was recorded in CHF CP-6 (229.30 cm²). The minimum number days taken for 50% flowering was found in genotypes CHF CP-9 (45.67).  &nbsp

Morphological Characterization of Underutilized Legume Sword Bean (Canavalia gladiata Jacq.) Genotypes of North-Eastern Hill Region of India Under Foot Hill of Arunachal Pradesh

Leguminous crops are important components of Indian horticulture, because of being an important source of nutrition in predominantly vegetarian diet. In order to investigate the genetic diversity present in the underutilized legume sword bean genotypes of North-Eastern hill region of India 20 native sword bean (Canavalia gladiata Jacq.)  genotypes were collected from different agroclimatic region of North-Eastern hill region of India. The morphological characters were observed in days to seed germination i.e. (3-6 days), pod length (16.45-32.87 cm), pod width (2.28-4.33 cm), pod weight (17.92-31.93 g), pod girth (7.75-12.64 mm) and seed weight (0.75-2.35 g). Performance of CHF SB-1, CHF SB-18, CHF SB-11, CHF SB-19, CHF SB-15, CHF SB-4 and CHF SB-10 genotypes under test conditions was superior and suggested that breeders might exploit the genome of these genotypes in current pulse crop improvement programmes

Comparison of kinetic parameters (<i>K_app</i> and <i>V_max</i>) of enzymatic activity of wild type OAS1 when activated by WNV SLI/II/III and its truncations.

<p>Comparison of kinetic parameters (<i>K_app</i> and <i>V_max</i>) of enzymatic activity of wild type OAS1 when activated by WNV SLI/II/III and its truncations.</p