Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Fast-Tracking Health Data Standards Development and Adoption in Real-World Settings: A Pilot Approach.

BACKGROUND: Pilot-testing is important in standards development because it facilitates agile navigation of the gap between needs for and use of standards in real-world settings and can reveal the practicalities of implementation. As the implementation and use of health data standards are usually more complicated than anticipated, the Office of the National Coordinator for Health Information Technology (ONC) routinely oversees and organizes relevant pilot projects. OBJECTIVES: This article provides an in-depth look into a sample of ONC's standards-focused pilot projects to (1) inform readers of the complexities of developing, implementing, and advancing standards and (2) guide those seeking to evaluate new standards through pilot projects. METHODS: The ONC's approach to conducting pilot projects begins with identifying a clinical care need, research requirement, or policy outcome that is not well supported by existing standards through a landscape review. ONC then selects a testing approach based on the identified need and maturity of relevant standards. Next, ONC identifies use cases and sites to pilot-test the relevant standard. Once complete, ONC publishes a report that informs subsequent projects and standards development. RESULTS: Pilot projects presented here are organized into three categories related to their demonstrated focus and related approach: (1) improving standards for presenting and sharing clinical genetic data, (2) accelerating the development and implementation of new standards, and (3) facilitating clinical data reuse. Each project illustrates the pilot approach from inception to next steps, capturing the role of collaboration among standards development organizations, stakeholders, and end-users to ensure standards are practical and fit for purpose. CONCLUSION: The ONC approach identifies implementation difficulties prior to broader adoption and use of standards, and provides insight into the steps needed to scale use of standards. The ONC's organization of pilot projects serves as a natural accelerator for building communities of practice, often providing a well-connected beneficiary of lessons learned.

Putative E3 ubiquitin ligase of human rotavirus inhibits NF-κB activation by using molecular mimicry to target ß-TrCP.

UNLABELLED: NF-κB plays a critical role in the induction and maintenance of innate and adaptive immune transcriptional programs. An associated inhibitor of κB protein (IκB) regulates NF-κB activation and contains a degron motif (DSGΦxS) that undergoes phosphorylation following pathogen recognition or other proinflammatory signals. The E3 ubiquitin ligase SCF(ß-TrCP) recognizes this phosphodegron through its ß-transducin repeat-containing protein (ß-TrCP) subunit and induces IκB degradation, allowing NF-κB to translocate to the nucleus and modulate gene expression. Rotavirus (RV), a major cause of pediatric gastroenteritis, can block NF-κB activation through the action of its nonstructural protein NSP1, a putative E3 ubiquitin ligase that mediates the degradation of ß-TrCP or other immunomodulatory proteins in a virus strain-specific manner. Here, we show that NSP1 targets ß-TrCP by mimicking the IκB phosphodegron. The NSP1 proteins of most human and porcine RV strains conserve a C-terminal phosphodegron-like (PDL) motif, DSGΦS. Deletion of this motif or mutation of its serine residues disrupts NSP1-mediated degradation of ß-TrCP and inhibition of NF-κB activation. Additionally, a point mutation within the phosphodegron-binding pocket protects ß-TrCP from NSP1-mediated turnover. Fusion of the PDL motif to an NSP1 protein known to target other immunomodulatory proteins generates a chimeric NSP1 protein that can induce ß-TrCP degradation and block NF-κB activation. Other viral proteins (Epstein-Barr virus LMP1, HIV-1 Vpu, and vaccinia virus A49) also contain a PDL motif and interact with ß-TrCP to inhibit NF-κB activation. Taken together, these data suggest that targeting ß-TrCP by molecular mimicry may be a common strategy used by human viruses to evade the host immune response. IMPORTANCE: The transcription factor NF-κB, a central regulator of the host response to infection, is a frequent target of viral antagonism. Pathogen detection activates NF-κB by inducing the phosphorylation of an associated inhibitor protein (IκB), which targets IκB for degradation by the E3 ubiquitin ligase ß-TrCP. Rotavirus, a significant cause of childhood gastroenteritis, antagonizes NF-κB through the activity of its NSP1 protein, a putative E3 ubiquitin ligase that mediates ß-TrCP turnover. Here, we show that NSP1 functions by mimicking the IκB phosphodegron recognized by ß-TrCP. Nearly all human rotavirus strains conserve this motif at the NSP1 C terminus, and its removal disrupts NSP1 antagonist activity. This sequence conserves the biochemical properties of the IκB phosphodegron and can rescue antagonist activity when fused to an NSP1 protein otherwise inactive against ß-TrCP. Other viral proteins also mimic IκB to disrupt NF-κB activation, indicating that this is an important immune evasion strategy.

Analysis of human rotaviruses from a single location over an 18-year time span suggests that protein coadaption influences gene constellations.

UNLABELLED: Rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses that cause severe gastroenteritis in children. In addition to an error-prone genome replication mechanism, RVs can increase their genetic diversity by reassorting genes during host coinfection. Such exchanges allow RVs to acquire advantageous genes and adapt in the face of selective pressures. However, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, coadaptive mutations and that operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P[8]-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years 1974 to 1991. Intragenotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs cocirculated during the vast majority of the collection years and that some of these GCs persisted in the community unchanged by reassortment. To investigate the influence of protein coadaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive covariance network. Unexpectedly, amino acid covariation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally coadapted, albeit noninteracting, viral proteins. This work raises important questions about mutation-reassortment interplay and its impact on human RV evolution. IMPORTANCE: Rotaviruses are devastating human pathogens that cause severe diarrhea and kill >450,000 children each year. The virus can evolve by accumulating mutations and by acquiring new genes from other strains via a process called reassortment. However, little is known about the relationship between mutation accumulation and gene reassortment for rotaviruses and how it impacts viral evolution. In this study, we analyzed the genome sequences of human strains found in clinical fecal specimens that were collected at a single hospital over an 18-year time span. We found that many rotaviruses did not reassort their genes but instead maintained them as specific sets (i.e., constellations). By analyzing the encoded proteins, we discovered concurrent amino acid changes among them, which suggests that they are functionally coadapted to operate best when kept together. This study increases our understanding of how rotaviruses evolve over time in the human population.

Absence of genetic differences among G10P[11] rotaviruses associated with asymptomatic and symptomatic neonatal infections in Vellore, India.

UNLABELLED: Rotaviruses (RVs) are leading causes of severe diarrhea and vomiting in infants and young children. RVs with G10P[11] genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants responsible for differences in symptomology, the genome sequences of G10P[11] RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as those of the previously characterized symptomatic N155 Vellore isolate. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P[11] viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic infections and from neonates with asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P[11] RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms. IMPORTANCE: Rotavirus (RV) strains have been identified that preferentially replicate in neonates, in some cases, without causing gastrointestinal disease. Surveillance studies have established that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, with half of infected neonates exhibiting symptoms. We used Sanger and next-generation sequencing technologies to contrast G10P[11] RVs recovered from symptomatic and asymptomatic neonates. Remarkably, the data showed that the RNA genomes of the viruses were virtually indistinguishable and lacked any differences that could explain the diversity of clinical outcomes among infected Vellore neonates. The sequencing results also indicated that some symptomatic and some asymptomatic Vellore neonates were infected with other enteric viruses (Aichi virus, astrovirus, salvirus/klassevirus); however, none could be correlated with the presence of symptoms in neonates. Together, our findings suggest that other poorly defined factors, not connected to the genetic makeup of the Vellore G10P[11] viruses, influence whether neonates develop gastrointestinal disease symptoms.

Predicted structure and domain organization of rotavirus capping enzyme and innate immune antagonist VP3.

UNLABELLED: Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3. IMPORTANCE: Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.

Molecular epidemiology of contemporary G2P[4] human rotaviruses cocirculating in a single U.S. community: footprints of a globally transitioning genotype.

UNLABELLED: Group A rotaviruses (RVs) remain a leading cause of childhood gastroenteritis worldwide. Although the G/P types of locally circulating RVs can vary from year to year and differ depending upon geographical location, those with G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8] specificities typically dominate. Little is known about the evolution and diversity of G2P[4] RVs and the possible role that widespread vaccine use has had on their increased frequency of detection. To address these issues, we analyzed the 12 G2P[4] RV isolates associated with a rise in RV gastroenteritis cases at Vanderbilt University Medical Center (VUMC) during the 2010-2011 winter season. Full-genome sequencing revealed that the isolates had genotype 2 constellations typical of DS-1-like viruses (G2P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2). Phylogenetic analyses showed that the genome segments of the isolates were comprised of two or three different subgenotype alleles; this enabled recognition of three distinct clades of G2P[4] viruses that caused disease at VUMC in the 2010-2011 season. Although the three clades cocirculated in the same community, there was no evidence of interclade reassortment. Bayesian analysis of 328 VP7 genes of G2 viruses isolated in the last 39 years indicate that existing G2 VP7 gene lineages continue to evolve and that novel lineages, as represented by the VUMC isolates, are constantly being formed. Moreover, G2 lineages are characteristically shaped by lineage turnover events that introduce new globally dominant strains every 7 years, on average. The ongoing evolution of G2 VP7 lineages may give rise to antigenic changes that undermine vaccine effectiveness in the long term. IMPORTANCE: Little is known about the diversity of cocirculating G2 rotaviruses and how their evolution may undermine the effectiveness of rotavirus vaccines. To expand our understanding of the potential genetic range exhibited by rotaviruses circulating in postvaccine communities, we analyzed part of a collection of rotaviruses recovered from pediatric patients in the United States from 2010 to 2011. Examining the genetic makeup of these viruses revealed they represented three segregated groups that did not exchange genetic material. The distinction between these three groups may be explained by three separate introductions. By comparing a specific gene, namely, VP7, of the recent rotavirus isolates to those from a collection recovered from U.S. children between 1974 and 1991 and other globally circulating rotaviruses, we were able to reconstruct the timing of events that shaped their ancestry. This analysis indicates that G2 rotaviruses are continuously evolving, accumulating changes in their genetic material as they infect new patients.