Complete genome sequence of a Pseudomonas fluorescens bacteriophage UNO-G1W1 isolated from freshwater ice in Nebraska.

We provide the complete genome sequence for a novel Pseudomonas fluorescens bacteriophage named UNO-G1W1. This phage was isolated from a single ice cover sampling. The genome was sequenced on the Nanopore MinION, generated with the direct terminal repeat-phage-pipeline and polished with Illumina short reads. Sequence identity classifies the phage as an otagovirus.

Role of RNA Domain Structure and Orientation in the Coxsackievirus B3 Virulence Phenotype.

Coxsackievirus B3 (CVB3) is an enterovirus that causes diseases such as pancreatitis and myocarditis in humans. Approximately 10% of the CVB3 RNA genome consists of a highly structured 5' untranslated region (5' UTR) that is organized into six domains and contains a type I internal ribosome entry site (IRES). These features are common to all enteroviruses. Each RNA domain plays a vital role in translation and replication during the viral multiplication cycle. We used SHAPE-MaP chemistry to generate secondary structures of the 5' UTR from the avirulent strain CVB3/GA and the virulent strain CVB3/28. Our comparative models show how key nucleotide substitutions cause major restructuring of domains II and III of the 5' UTR in CVB3/GA. Despite these structural shifts, the molecule maintains several well-characterized RNA elements, which allows persistence of the unique avirulent strain. The results shed light on the 5' UTR regions serving as virulence determinants and those required for fundamental viral mechanisms. We used the SHAPE-MaP data to produce theoretical tertiary models using 3dRNA v2.0. These models suggest a compact conformation of the 5' UTR from the virulent strain CVB3/28 that brings critical domains into close contact. In contrast, the model of the 5' UTR from the avirulent strain CVB3/GA suggests a more extended conformation where the same critical domains are more separated. Our results suggest that the structure and orientation of RNA domains in the 5' UTR are responsible for low-efficiency translation, low viral titers, and absence of virulence observed during infection by CVB3/GA. IMPORTANCE Human enteroviruses, which include five different species and over 100 serotypes, are responsible for diseases ranging from mild respiratory infections to serious infections of pancreas, heart, and neural tissue. All enteroviral RNA genomes have a long and highly structured 5' untranslated region (5' UTR) containing an internal ribosome entry site (IRES). Major virulence determinants are located in the 5' UTR. We present RNA structure models that directly compare the 5' UTR derived from virulent and avirulent strains of the enterovirus coxsackievirus B3 (CVB3). The secondary-structure models show rearrangement of RNA domains known to be virulence determinants and conservation of structure in RNA elements known to be vital for translation and replication in the avirulent strain CVB3/GA. The tertiary-structure models reveal reorientation of RNA domains in CVB3/GA. Identifying the details of structure in these critical RNA domains will help direct antiviral approaches to this major human pathogen.

RNA Structure in the 5' Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes.

Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5' UTR of the genome compared to the historical Fermon strain. The structure of the 5' UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5' UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5' UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.

An instructional definition and assessment rubric for bioinformatics instruction.

The lack of an instructional definition of bioinformatics delays its effective integration into biology coursework. Using an iterative process, our team of biologists, a mathematician/computer scientist, and a bioinformatician together with an educational evaluation and assessment specialist, developed an instructional definition of the discipline: Bioinformatics is "an interdisciplinary field that is concerned with the development and application of algorithms that analyze biological data to investigate the structure and function of biological polymers and their relationships to living systems." The field is defined in terms of its two primary foundational disciplines, biology and computer science, and its interdisciplinary nature. At the same time, we also created a rubric for assessing open-ended responses to a prompt about what bioinformatics is and how it is used. The rubric has been shown to be reliable in successive rounds of testing using both common percent agreement (89.7%) and intraclass correlation coefficient (0.620) calculations. We offer the definition and rubric to life sciences instructors to help further integrate bioinformatics into biology instruction, as well as for fostering further educational research projects.

Incubators: Building community networks and developing open educational resources to integrate bioinformatics into life science education.

While it is essential for life science students to be trained in modern techniques and approaches, rapidly developing, interdisciplinary fields such as bioinformatics present distinct challenges to undergraduate educators. In particular, many educators lack training in new fields, and high-quality teaching and learning materials may be sparse. To address this challenge with respect to bioinformatics, the Network for the Integration of Bioinformatics into Life Science Education (NIBLSE), in partnership with Quantitative Undergraduate Biology Education and Synthesis (QUBES), developed incubators, a novel collaborative process for the development of open educational resources (OER). Incubators are short-term, online communities that refine unpublished teaching lessons into more polished and widely usable learning resources. The resulting products are published and made freely available in the NIBLSE Resource Collection, providing recognition of scholarly work by incubator participants. In addition to producing accessible, high-quality resources, incubators also provide opportunities for faculty development. Because participants are intentionally chosen to represent a range of expertise in bioinformatics and pedagogy, incubators also build professional connections among educators with diverse backgrounds and perspectives and promote the discussion of practical issues involved in deploying a resource in the classroom. Here we describe the incubator process and provide examples of beneficial outcomes. Our experience indicates that incubators are a low cost, short-term, flexible method for the development of OERs and professional community that could be adapted to a variety of disciplinary and pedagogical contexts.

Structure of the 5' Untranslated Region of Enteroviral Genomic RNA.

Enteroviral RNA genomes share a long, highly structured 5' untranslated region (5' UTR) containing a type I internal ribosome entry site (IRES). The 5' UTR is composed of stably folded RNA domains connected by unstructured RNA regions. Proper folding and functioning of the 5' UTR underlies the efficiency of viral replication and also determines viral virulence. We have characterized the structure of 5' UTR genomic RNA from coxsackievirus B3 using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and base-specific chemical probes in solution. Our results revealed novel structural features, including realignment of major domains, newly identified long-range interactions, and an intrinsically disordered connecting region. Together, these newly identified features contribute to a model for enteroviral 5' UTRs with type I IRES elements that links structure to function during the hierarchical processes directed by genomic RNA during viral infection.IMPORTANCE Enterovirus infections are responsible for human diseases, including myocarditis, pancreatitis, acute flaccid paralysis, and poliomyelitis. The virulence of these viruses depends on efficient recognition of the RNA genome by a large family of host proteins and protein synthesis factors, which in turn relies on the three-dimensional folding of the first 750 nucleotides of the molecule. Structural information about this region of the genome, called the 5' untranslated region (5' UTR), is needed to assist in the process of vaccine and antiviral development. This work presents a model for the structure of the enteroviral 5' UTR. The model includes an RNA element called an intrinsically disordered RNA region (IDRR). Intrinsically disordered proteins (IDPs) are well known, but correlates in RNA have not been proposed. The proposed IDRR is a 20-nucleotide region, long known for its functional importance, where structural flexibility helps explain recognition by factors controlling multiple functional states.

Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an avirulent strain.

Coxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5' untranslated region (5'UTR) to complete critical viral processes. Here, we directly compared the structure of a 5'UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5'UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5'UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104-184 and repeating the 5'UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5'UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5'UTR lacks structure in the SLII cardiovirulence determinant.

Structure of the 5' nontranslated region of the coxsackievirus b3 genome: Chemical modification and comparative sequence analysis.

Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5' end of the viral genome contains a highly structured nontranslated region (5'NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5'NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5'NTR. Chemical probing results show that the theoretical model for the CVB3 5'NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses.

Mechanism of gonadotropin gene expression. Identification of a novel negative regulatory element at the transcription start site of the glycoprotein hormone alpha-subunit gene.

Regulation of the glycoprotein hormone alpha-subunit (GPHalpha) gene has been studied extensively in pituitary and placental cell lines, but little is known of the transcriptional regulators important for its ectopic expression. To investigate the molecular basis for ectopic expression, it was critical to define cis-regulatory elements and their cognate trans-acting factors that modulate promoter activity in epithelial cell types that do not normally express GPH. DNA-mediated transient expression of promoter-reporter constructs was used to identify a novel negative regulatory element located at the GPHalpha gene transcription start site. Truncation or site-directed mutagenesis of this element produced up to a 10-fold increase in promoter activity. Electrophoretic mobility shift analysis detected a protein that binds specifically to a DNA motif encompassing the cap site. Based on competitive DNA binding studies with mutated oligonucleotides, it was determined that bases from -5 to -2 and +4 to +11 are critical for protein binding. The DNA sequence flanking the transcription start site from -9 to +11 is an imperfect palindrome; consequently, this motif is referred to as the cap site diad element (CSDE) and the cognate factor as the cap site-binding protein (CSBP). CSBP activity was present at different levels in nuclear extracts prepared from a variety of cell types. Significantly, the ratio of activities exhibited by the GPHalpha promoter with a mutated CSDE compared with the promoter with a wild-type CSDE was dependent on the transfected cell line and its content of CSBP. These results indicate that a negative regulatory element centered at the GPHalpha gene cap site and its cognate DNA-binding protein make a significant contribution to the production of alpha-subunit in a variety of tumor tissues. A detailed understanding of this cis/trans pair may further suggest a mechanism to explain, at least in part, how this gene becomes activated in nonendocrine tumors.