Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5 termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5 CG or 5 AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.The six serotypes of the group B coxsackieviruses (CVB1-6) are enteroviruses (Picornaviridae, species HEV-B) (53). The CVB genome is a single-stranded RNA molecule, 7,400 nucleotides (nt) in length, that is encapsidated within an icosahedral shell (74). The 11 viral proteins (83) are encoded by a single open reading frame which is flanked on the 5Ј and 3Ј termini by nontranslated regions (NTRs) (20). The CVB induce numerous human illnesses, including inflammatory heart disease, pancreatitis, and aseptic meningitis and may also trigger the onset of type 1 diabetes (5,31,72,81,82). The CVB were recognized as causes of human heart disease shortly after their description early in the 1950s (26, 27) and remain the enteroviruses most commonly associated with human cardiomyopathies on the grounds of isolation and serology (5,6,34,62,88). Meta-analysis shows an association between enteroviruses and myocarditis in 23% of cases (7), although this association is more variable in cases of dilated cardiomyopathy, a serious disease that often leads to a failing heart (61). Mouse models of CVB-induced pancreatitis (94, 110), myocarditis (45,52,110), myositis (104, 105), and rapid-onset type 1 diabetes (31) which facilitate the study of these diseases have been developed.Enterovirus infections are generally considered to be acute events, with symptoms and virus titers peaking within a few days postinoculation (p.i.) and with virus being cleared by the adaptive immune response (17). However, enterovirus infections can persist under conditions of immunodeficiency (48,51,...

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 (5NTR) 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 5NTR, 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 5NTR. Chemical probing results show that the theoretical model for the CVB3 5NTR 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.Coxsackievirus B3 (CVB3) is a member of the Enterovirus genus of the family Picornaviridae. As with all picornaviruses, the CVB3 genome is a single-stranded positive-sense RNA that is organized into four sections: a highly structured 5Ј nontranslated region (5ЈNTR), a single open reading frame encoding a polyprotein, a 3ЈNTR, and a poly(A) tail (48). For CVB3 the 7,400-nucleotide genome is composed of a 5ЈNTR of approximately 742 bases, a coding region that specifies a 2,185-amino-acid polyprotein, a 98-nucleotide (nt) 3ЈNTR, and a poly(A) tail (30,36). Upon entering a permissive host cell, the enterovirus genome first serves as a template for translation, producing the viral polyprotein, and then becomes a template for replication of the minus strand. Both of these functions, as well as the regulatory switch between them, require critical structural elements in the 5ЈNTR RNA (18,19,45,52). Alteration of structural elements in the 5ЈNTR severely compromises viral multiplication and also abrogates virulence (6).The enterovirus cap-independent translation mechanism has been studied extensively, particularly in poliovirus, and serves as a model for all enteroviruses, including CVB3. In this translation mechanism, the 5ЈNTR contains a cis-acting internal ribosome entry site (IRES) (26,50,60) that recruits ribosomes directly to a downstream AUG codon, thereby circu...

Although bioinformatics is becoming increasingly central to research in the life sciences, bioinformatics skills and knowledge are not well integrated into undergraduate biology education. This curricular gap prevents biology students from harnessing the full potential of their education, limiting their career opportunities and slowing research innovation. To advance the integration of bioinformatics into life sciences education, a framework of core bioinformatics competencies is needed. To that end, we here report the results of a survey of biology faculty in the United States about teaching bioinformatics to undergraduate life scientists. Responses were received from 1,260 faculty representing institutions in all fifty states with a combined capacity to educate hundreds of thousands of students every year. Results indicate strong, widespread agreement that bioinformatics knowledge and skills are critical for undergraduate life scientists as well as considerable agreement about which skills are necessary. Perceptions of the importance of some skills varied with the respondent’s degree of training, time since degree earned, and/or the Carnegie Classification of the respondent’s institution. To assess which skills are currently being taught, we analyzed syllabi of courses with bioinformatics content submitted by survey respondents. Finally, we used the survey results, the analysis of the syllabi, and our collective research and teaching expertise to develop a set of bioinformatics core competencies for undergraduate biology students. These core competencies are intended to serve as a guide for institutions as they work to integrate bioinformatics into their life sciences curricula.

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia colimrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affiity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.Several regions of rRNA have been shown to be involved in the process of translation, primarily regions that are single stranded and highly conserved phylogenetically (1,2). The locations of these sequences in Escherichia coli 16S rRNA have now been placed in three-dimensional models ofthe 30S ribosomal subunit (3, 4). In general there is good agreement between structure and function since many single-stranded highly conserved rRNA regions, proposed to carry out related functions in translation, are clustered in the same area of the subunit (5).One example of a region with a structural placement consistent with its proposed function is the 790 loop of 16S rRNA. In this 9-base loop in the central domain of 16S rRNA, 6 of the bases are universally conserved and 2 additional bases are strongly but not universally conserved (6). The loop has been localized to the platform of the subunit in the area that interfaces with the 50S subunit (3, 4, 7). Several studies have established that many of the bases in the 790 loop are exposed in 30S subunits and inaccessible in 70S ribosomes (8-13). Some of these same studies have implicated this region in the process of subunit association (9-11).The chemical modification studies by Moazed and Noller (13,14), in which rRNA accessibilities were probed in the presence of tRNA or antibiotics, showed that the 790 loop influences several aspects of protein synthesis aside from subunit association. To further define its functional role, the universally conserved guanine at position 791 was mutated to an adenine by site-directed mutagenesis. Here we report on the structural and functional characteristics of the resulting mutant 30S subunits. MATERIALS AND METHODSMutagenesis and Expression. The single-base mutation at position 791 was constructed by oligonucleotide-directed mutagenesis in M13 as described by Zoller and Smith (15).The EcoRI-Xba I fragment from rrnB was cloned into M13mpl9 and the template was isolated from the ung-dutstrain RZ1032 to increase the frequency of mutagenesis (16). After mutagenesis M13 isolates were screened by DNA sequencing (17) and the Bgl II-Xba I fragment containing the mutation was cloned into the expression vector pKK353...