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

Lentil anthracnose: epidemiology, fungicide decision support system, resistance and pathogen races

<p><i>Colletotrichum lentis</i> causes anthracnose of lentil (<i>Lens culinaris</i>) in Canada that results in defoliation, stem girdling and severe yield losses, but the disease is rarely reported elsewhere. The pathogen survives as microsclerotia on lentil debris for up to 3 years when buried in the soil, but loses viability on the soil surface. Windborne debris spreads the pathogen to neighbouring fields, while seedborne infection is less important. Foliar fungicides were registered, and a fungicide decision support system was developed which assessed disease risk with 85% accuracy. Around 2300 <i>L. culinaris</i> accessions from 50 countries were screened for resistance. Congruently, two races – Ct1 and Ct0 – were identified on differential lentil lines. Resistant lines were generated by cycles of inoculation and selfing of single resistant plants which resulted in the following three accessions resistant to both races: VIR2633 (Georgia), VIR2058 and VIR2076 (Czech Republic), while six and 49 lines had resistance to Ct0 and Ct1, respectively. Ct1 resistance is controlled by recessive and dominant genes <i>crt1</i> and <i>CtR3</i> in variety ‘Indianhead’, <i>ctr2</i> and <i>CtR5</i> in accession PI345629 and <i>CtR4</i> in PI320937. Molecular markers linked to Ct1 resistance were identified on linkage group six, close to <i>Ascochyta lentis</i> resistance, and were used to combine resistance to both pathogens in breeding lines. Two repeat rich regions in the intergenic spacer (IGS) of ribosomal DNA can be used to differentiate the two <i>C. lentis</i> races. Utilizing length polymorphisms in a 39 nucleotide repeat region showed the races were equally frequent among isolates collected between 1991 and 1999, while 95% belonged to race Ct0 in 2010, likely because lentil varieties are susceptible to race Ct0, but around one-third of the varieties had Ct1 resistance.</p

The Dynamic Landscape of the Full-Length HIV‑1 Transactivator of Transcription

The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales. Sequential backbone assignment was facilitated by several isotope labeling schemes, including uniform labeling, site-specific labeling, and unlabeling. ¹⁵N NMR relaxation parameters were measured and analyzed by reduced spectral density mapping and the Lipari–Szabo Model-Free approach to characterize the backbone dynamics on the picosecond to nanosecond time scale. The results indicate that the protein exists in an extended disordered conformational ensemble. NMR relaxation dispersion profiles show that on the millisecond time scale no conformational exchange is detected for any of the residues, supporting the model of a disordered backbone. NMR chemical shift differences from random coil values suggest that some segments of the protein have a modest propensity to fold; comparison to X-ray diffraction structures of Tat complexes indicates that some segments of the protein function through an induced-fit mechanism whereas other segments likely operate by conformational selection. Surprisingly, measured hydrogen exchange rates are higher than predicted for a disordered polymer, but this is explained as being caused by the high net charge on the protein that enhances base-catalyzed hydrogen exchange. The dynamics results provide a deeper understanding of the protein conformational ensemble and form a foundation for future studies of the conformational changes that accompany the formation of the superelongation complex that activates viral transcription

Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element) is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes

Recombinant human OAS1 adopts a globular fold.

<p>(<b>A</b>) Sedimentation velocity (SV) distribution analysis in terms of <i>c</i>(S) at 0.4 mg/mL. In-set is the resultant concentration dependence of the SV distribution. (<b>B</b>) Concentration dependence of hydrodynamic radius obtained from DLS measurements. (<b>C</b>) The pair distribution function versus particle radius obtained from the GNOM analysis. In-set is the merged scattering data obtained from multiple concentrations. (<b>D</b>) Superimposition of the human OAS1 (PDB 4IG8) high-resolution structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092545#pone.0092545-Donovan1" target="_blank">[26]</a> on the <i>ab initio</i> model generated using DAMMIF on the data obtained from SAXS experiments on human OAS1.</p

Experimental and predicted hydrodynamic parameters of OAS1 and SLI/II/III (error shown in parentheses).

a<p>experimentally determined from DLS data.</p>b<p>from AUC-SV data.</p>c<p>from SAXS data.</p>d<p>the <i>r_G</i> values for R195E and K199E are 2.43 (0.11) nm and 2.40 (0.13) nm respectively.</p>e<p>the <i>D_max</i> values for R195E and K199E are 6.9 nm and 7.0 nm respectively.</p>f<p>based on homology with high-resolution structure of human OAS1.</p

The WNV SLI/II/III forms a direct interaction with human OAS1.

<p>(<b>A</b>) EMSA for OAS1 (100 nM) binding to the SLI/II/III under non-denaturing conditions. (<b>B</b>) EMSA for OAS1 (100 nM) binding to SLI+II under non-denaturing conditions. (<b>C</b>) Non-denaturing gel electrophoresis of SLI/II/III truncations (100 nM) in the presence or absence of OAS1 (400 nM). In all cases, 8% native TBE gels were used and stained with Sybr Gold (Invitrogen, USA) to visualize RNA-containing species.</p

Comparison of kinetic parameters (<i>K_app</i> and <i>V_max</i>) of enzymatic activity of wild type and mutant OAS1s when activated by the WNV SLI/II/III.

<p>Comparison of kinetic parameters (<i>K_app</i> and <i>V_max</i>) of enzymatic activity of wild type and mutant OAS1s when activated by the WNV SLI/II/III.</p