David J. Esteban

A Poxvirus Protein That Binds to and Inactivates IL-18, and Inhibits NK Cell Response

IL-18 induces IFN-γ and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response.

Adenovirus RIDβ Subunit Contains a Tyrosine Residue That Is Critical for RID-Mediated Receptor Internalization and Inhibition of Fas- and TRAIL-Induced Apoptosis

ABSTRACT The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDα and RIDβ subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these “death” receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDβ that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDβ were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDβ C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxφ). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDβ but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RIDs ability to down-regulate receptors.

Genomic sequence and analysis of a vaccinia virus isolate from a patient with a smallpox vaccine-related complication.

BackgroundVaccinia virus (VACV)-DUKE was isolated from a lesion on a 54 year old female who presented to a doctor at the Duke University Medical Center. She was diagnosed with progressive vaccinia and treated with vaccinia immune globulin. The availability of the VACV-DUKE genome sequence permits a first time genomic comparison of a VACV isolate associated with a smallpox vaccine complication with the sequence of culture-derived clonal isolates of the Dryvax vaccine.ResultsThis study showed that VACV-DUKE is most similar to VACV-ACAM2000 and CLONE3, two VACV clones isolated from the Dryvax® vaccine stock confirming VACV-DUKE as an isolate from Dryvax®. However, VACV-DUKE is unique because it is, to date, the only Dryvax® clone isolated from a patient experiencing a vaccine-associated complication. The 199,960 bp VACV-DUKE genome encodes 225 open reading frames, including 178 intact genes and 47 gene fragments. Between VACV-DUKE and the other Dryvax® isolates, the major genomic differences are in fragmentation of the ankyrin-like, and kelch-like genes, presence of a full-length Interferon-α/β receptor gene, and the absence of a duplication of 12 ORFs in the inverted terminal repeat. Excluding this region, the DNA sequence of VACV-DUKE differs from the other two Dryvax® isolates by less than 0.4%. DNA sequencing also indicated that there was little heterogeneity in the sample, supporting the hypothesis that virus from an individual lesion is clonal in origin despite the fact that the vaccine is a mixed population.ConclusionVirus in lesions that result from progressive vaccinia following vaccination with Dryvax are likely clonal in origin. The genomic sequence of VACV-DUKE is overall very similar to that of Dryvax® cell culture-derived clonal isolates. Furthermore, with the sequences of multiple clones from Dryvax® we can begin to appreciate the diversity of the viral population in the smallpox vaccine.

Mousepox, A Small Animal Model of Smallpox

Ectromelia virus infections in the laboratory mouse have emerged as a valuable model to investigate human orthopoxvirus infections to understand the progression of disease, to discover and characterize antiviral treatments, and to study the host-pathogen relationship as it relates to pathogenesis and the immune response. Here we describe how to safely work with the virus and protocols for common procedures for the study of ectromelia virus in the laboratory mouse including the preparation of virus stocks, the use of various routes of inoculation, and collection of blood and tissue from infected animals. In addition, several procedures are described for assessing the host response to infection: for example, measurement of virus-specific CD8 T cells and the use of ELISA and neutralization assays to measure orthopoxvirus-specific antibody titers.

New bioinformatics tools for viral genome analyses at Viral Bioinformatics - Canada

Viruses are much smaller than prokaryotes and eukaryotes, and it is now practical to sequence closely related members of virus families, strains, or even different isolates recovered during the course of an outbreak. However, comparative analysis of viral genomes requires the development of novel bioinformatics tools that allow us to align, edit, compare and interact with these genomes at all levels, from whole genome, to gene family, to single nucleotide polymorphisms. Comparative viral genomics can lead to the identification of the core characteristics that define a virus family, as well as the unique properties of viral species or isolates that contribute to variations in pathogenesis. This paper describes a number of tools, mainly developed for Viral Bioinformatics--Canada, that can be used for annotation and comparative genomic analysis of poxviruses. Nonetheless, these tools are also broadly applicable to other virus families.

Integrating Genomics Research throughout the Undergraduate Curriculum: A Collection of Inquiry-Based Genomics Lab Modules

We wish to let CBE—Life Sciences Education readers know about a portal to a set of curricular lab modules designed to integrate genomics and bioinformatics into commonly taught courses at all levels of the undergraduate curriculum. Through a multi-year, collaborative process, we developed, implemented, and peer-reviewed inquiry-based, integrated instructional units (I3Us) adaptable to a range of teaching settings, with a focus on both model and nonmodel systems. Each of the products is built on vetted design principles: 1) they have clear pedagogical objectives; 2) they are integrated with lessons taught in the lecture; 3) they are designed to integrate the learning of science content with learning about the process of science; and 4) they require student reflection and discussion (Figure 1; National Research Council [NRC], 2005). Eleven I3Us were designed and implemented as multi-week modules within the context of an existing biology course (e.g., microbiology, comparative anatomy, introduction to neurobiology), and three I3Us were incorporated into interdisciplinary biology/computer science classes. Our collection of genomics instructional units, together with extensive supporting material for each module, is accessible on a dedicated website (http://serc.carleton.edu/genomics/activities.html) that also provides links to bioinformatics tools and online assessment and pedagogical resources for teaching genomics. n n n nFigure 1. n nPedagogical elements of the I3U, which was based on the findings of Americas Lab Report (NRC, 2005 ) and was used as the primary curricular design framework for this project. n n n nRapid advances in genome sequencing and analysis offer unparalleled opportunity and challenge for biology educators. More data are being generated than can be analyzed and contextualized in traditional teaching or research models. Indeed, this explosion of data has spawned rapid growth in the discipline of bioinformatics, which is focused on development of the computational tools and approaches for extracting biologically meaningful insights from genomic data. At the same time, access to vast quantities of genomic data stored in publicly available databases can offer educators ways to engage undergraduates in authentic research and to democratize research that was previously possible only at research-intensive universities with massive instrumentation infrastructures. The integration of genomic and bioinformatic approaches into undergraduate curricula represents one response to the national calls for biology teaching that is more quantitative and promotes deeper understanding of biological systems through interdisciplinary analyses (National Academy of Sciences, 2003 ; Association of American Medical Colleges and Howard Hughes Medical Institute [HHMI], 2009 ; NRC, 2009 ; American Association for the Advancement of Science, 2011 ). Yet relatively few faculty members who teach undergraduate biology have expertise in the fields of genomics or bioinformatics, and they may therefore feel inadequately prepared to develop their own new curricular modules capitalizing on this dispersed abundance of available resources. n nOur Teagle Foundation–funded genomics education initiative, Bringing Big Science to Small Colleges: A Genomics Collaboration, was designed to address the challenges of helping faculty members integrate genome-scale science into the undergraduate classroom. The goal of the project was to create and disseminate self-contained curricular units that stimulate students and faculty alike to think in new ways and at different scales of biological inquiry. To this end, a series of three workshops over 3 yr brought together a total of 34 faculty participants from 19 institutions and a diverse array of disciplines—including biology, computer science, and science education—to develop a set of lab modules containing a substantial genomics component. We believe that these modules are suitable for integration into existing courses in the biology curriculum and are adaptable to a variety of teaching settings. n nThe project website serves as a portal to activity sheets describing each I3U, complete with learning goals, teaching tips, and links to teaching materials, as well as downloadable resources and assessment tools (Figure 2), that can be customized by any interested educator. Each I3U was peer-reviewed by fellow participants, as well as by a professional project consultant who has extensive experience with Web-based description of teaching materials using this format (Manduca et al., 2006 ). The goals of this review process were to ensure that each I3U met the design criteria articulated above, and to evaluate whether the activity sheet provided both an easily accessible overview of the content and enough detailed information for other instructors to adapt and implement the material and its associated assessment strategies. This peer review was complemented by each participants own explicitly framed evaluation of his/her I3U through a formal reflection form (accessible at http://serc.carleton.edu/genomics/workshop09/index.html). Although these I3Us were designed for courses currently taught by the project participants within the specific institutions’ curricula, we propose that they can be inserted into other courses encompassing similar content (Tables 1 and u200band2)2) and/or learning goals (e.g., Figure 2). We have received many communications from colleagues at other institutions who have adapted our I3Us for their courses. n n n nFigure 2. n nExcerpt from an activity sheet from the Genomics Instructional Units Minicollection describing one of the curricular modules developed within the Bringing Big Science to Small Colleges program (for the complete activity sheet, see http://serc.carleton.edu/genomics/units/19163.html ... n n n n n nTable 1. n nList of I3Us generated in the Bringing Big Science to Small Colleges collaborative project, grouped by the general level in the curriculum in which they were originally taught n n n n n nTable 2. n nPedagogical attributes (scale of biological organization, genomic level of analysis, and bioinformatic skills taught) of I3Us developed in this project and disseminated on the projects website n n n nOne fundamental characteristic of each I3U in our collection is the focus on guided inquiry. The benefits to an undergraduate of hands-on participation in research are well documented (Nagda et al., 1998 ; Gafney, 2001 ; Hunter et al., 2007 ; Kardash et al., 2008 ; Lopatto, 2009 ). Integrating authentic research experiences into the undergraduate curriculum allows this powerful learning model to be scaled from use with only a few students to use with entire laboratory sections (Lopatto 2009 ; Lopatto et al. 2008 ). Like other national participatory genomic teaching initiatives (Campbell et al., 2006 , 2007 ; Ditty et al., 2010 ; Shaffer et al., 2010 ; HHMI, 2011 ), our model for curriculum development in genomics emphasizes synergies between student-centered research and education. However, in contrast with some of these other projects, our grassroots approach leveraged a wealth of existing expertise by providing opportunities for individual faculty members to develop, implement, modify, evaluate, and share undergraduate teaching modules that stem from their own research and/or teaching interests. In this regard, our project most closely resembles the Genome Consortium for Active Teaching, which provides faculty members and their undergraduates access to microarrays from a variety of organisms, allowing participants to define their own research questions in a model system with which they are already familiar (Campbell et al., 2006 , 2007 ). n nOur collaborative effort among biologists, computer scientists, and science educators has yielded a collection of pedagogical resources that can be adapted for use in a wide variety of educational settings. We invite other biologists to begin building on our work by using and providing feedback on our I3Us. Faculty who have tested materials that exemplify our design principles are encouraged to add them to our collection. For further information, please contact the corresponding author.

Genes in the terminal regions of orthopoxvirus genomes experience adaptive molecular evolution

BackgroundOrthopoxviruses are dsDNA viruses with large genomes, some encoding over 200 genes. Genes essential for viral replication are located in the center of the linear genome and genes encoding host response modifiers and other host interacting proteins are located in the terminal regions. The central portion of the genome is highly conserved, both in gene content and sequence, while the terminal regions are more diverse. In this study, we investigated the role of adaptive molecular evolution in poxvirus genes and the selective pressures that act on the different regions of the genome. The relative fixation rates of synonymous and non-synonymous mutations (the dN/dS ratio) are an indicator of the mechanism of evolution of sequences, and can be used to identify purifying, neutral, or diversifying selection acting on a gene. Like highly conserved residues, amino acids under diversifying selection may be functionally important. Many genes experiencing diversifying selection are involved in host-pathogen interactions, such as antigen-antibody interactions, or the host-pathogen arms race.ResultsWe analyzed 175 gene families from orthopoxviruses for evidence of diversifying selection. 79 genes were identified as experiencing diversifying selection, 25 with high confidence. Many of these genes are located in the terminal regions of the genome and function to modify the host response to infection or are virion-associated, indicating a greater role for diversifying selection in host-interacting genes. Of the 79 genes, 20 are of unknown function, and implicating diversifying selection as an important mechanism in their evolution may help characterize their function or identify important functional residues.ConclusionsWe conclude that diversifying selection is an important mechanism of orthopoxvirus evolution. Diversifying selection in poxviruses may be the result of interaction with host defense mechanisms.

16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns

A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities.

Recent Hits Acquired by BLAST (ReHAB): A tool to identify new hits in sequence similarity searches

BackgroundSequence similarity searching is a powerful tool to help develop hypotheses in the quest to assign functional, structural and evolutionary information to DNA and protein sequences. As sequence databases continue to grow exponentially, it becomes increasingly important to repeat searches at frequent intervals, and similarity searches retrieve larger and larger sets of results. New and potentially significant results may be buried in a long list of previously obtained sequence hits from past searches.ResultsReHAB (Recent Hits Acquired from BLAST) is a tool for finding new protein hits in repeated PSI-BLAST searches. ReHAB compares results from PSI-BLAST searches performed with two versions of a protein sequence database and highlights hits that are present only in the updated database. Results are presented in an easily comprehended table, or in a BLAST-like report, using colors to highlight the new hits. ReHAB is designed to handle large numbers of query sequences, such as whole genomes or sets of genomes. Advanced computer skills are not needed to use ReHAB; the graphics interface is simple to use and was designed with the bench biologist in mind.ConclusionsThis software greatly simplifies the problem of evaluating the output of large numbers of protein database searches.

Temporal and Spatial Distribution of the Microbial Community of Winogradsky Columns.

Winogradsky columns are model microbial ecosystems prepared by adding pond sediment to a clear cylinder with additional supplements and incubated with light. Environmental gradients develop within the column creating diverse niches that allow enrichment of specific bacteria. The enrichment culture can be used to study soil and sediment microbial community structure and function. In this study we used a 16S rRNA gene survey to characterize the microbial community dynamics during Winogradsky column development to determine the rate and extent of change from the source sediment community. Over a period of 60 days, the microbial community changed from the founding pond sediment population: Cyanobacteria, Chloroflexi, Nitrospirae, and Planctomycetes increased in relative abundance over time, while most Proteobacteria decreased in relative abundance. A unique, light-dependent surface biofilm community formed by 60 days that was less diverse and dominated by a few highly abundant bacteria. 67–72% of the surface community was comprised of highly enriched taxa that were rare in the source pond sediment, including the Cyanobacteria Anabaena, a member of the Gemmatimonadetes phylum, and a member of the Chloroflexi class Anaerolinea. This indicates that rare taxa can become abundant under appropriate environmental conditions and supports the hypothesis that rare taxa serve as a microbial seed bank. We also present preliminary findings that suggest that bacteriophages may be active in the Winogradsky community. The dynamics of certain taxa, most notably the Cyanobacteria, showed a bloom-and-decline pattern, consistent with bacteriophage predation as predicted in the kill-the-winner hypothesis. Time-lapse photography also supported the possibility of bacteriophage activity, revealing a pattern of colony clearance similar to formation of viral plaques. The Winogradsky column, a technique developed early in the history of microbial ecology to enrich soil microbes, may therefore be a useful model system to investigate both microbial and viral ecology.