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1.  Genome Sequence of a Bornavirus Recovered from an African Garter Snake (Elapsoidea loveridgei) 
Genome Announcements  2014;2(5):e00779-14.
Bornaviruses are known to infect mammals and birds, and they have been associated with disease in both groups of animals. Here, we report the genome sequence of a bornavirus identified in a wild-caught Loveridge’s garter snake (Elapsoidea loveridgei).
doi:10.1128/genomeA.00779-14
PMCID: PMC4192372  PMID: 25301640
2.  Ball Python Nidovirus: a Candidate Etiologic Agent for Severe Respiratory Disease in Python regius 
mBio  2014;5(5):e01484-14.
ABSTRACT
A severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease.
IMPORTANCE
Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals.
doi:10.1128/mBio.01484-14
PMCID: PMC4173777  PMID: 25205093
3.  Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing 
The New England journal of medicine  2014;370(25):2408-2417.
SUMMARY
A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.
doi:10.1056/NEJMoa1401268
PMCID: PMC4134948  PMID: 24896819
4.  Transcriptome-wide characterization of the eIF4A signature highlights plasticity in translation regulation 
Genome Biology  2014;15(10):476.
Background
Protein synthesis is tightly regulated and alterations to translation are characteristic of many cancers. Translation regulation is largely exerted at initiation through the eukaryotic translation initiation factor 4 F (eIF4F). eIF4F is pivotal for oncogenic signaling as it integrates mitogenic signals to amplify production of pro-growth and pro-survival factors. Convergence of these signals on eIF4F positions this factor as a gatekeeper of malignant fate. While the oncogenic properties of eIF4F have been characterized, genome-wide evaluation of eIF4F translational output is incomplete yet critical for developing novel translation-targeted therapies.
Results
To understand the impact of eIF4F on malignancy, we utilized a genome-wide ribosome profiling approach to identify eIF4F-driven mRNAs in MDA-MB-231 breast cancer cells. Using Silvestrol, a selective eIF4A inhibitor, we identify 284 genes that rely on eIF4A for efficient translation. Our screen confirmed several known eIF4F-dependent genes and identified many unrecognized targets of translation regulation. We show that 5′UTR complexity determines Silvestrol-sensitivity and altering 5′UTR structure modifies translational output. We highlight physiological implications of eIF4A inhibition, providing mechanistic insight into eIF4F pro-oncogenic activity.
Conclusions
Here we describe the transcriptome-wide consequence of eIF4A inhibition in malignant cells, define mRNA features that confer eIF4A dependence, and provide genetic support for Silvestrol’s anti-oncogenic properties. Importantly, our results show that eIF4A inhibition alters translation of an mRNA subset distinct from those affected by mTOR-mediated eIF4E inhibition. These results have significant implications for therapeutically targeting translation and underscore a dynamic role for eIF4F in remodeling the proteome toward malignancy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0476-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0476-1
PMCID: PMC4203936  PMID: 25273840
5.  Structure of the Transcriptional Network Controlling White-Opaque Switching in Candida albicans 
Molecular microbiology  2013;90(1):22-35.
Summary
The human fungal pathogen Candida albicans can switch between two phenotypic cell types, termed “white” and “opaque.” Both cell types are heritable for many generations, and the switch between the two types occurs epigenetically, that is, without a change in the primary DNA sequence of the genome. Previous work identified six key transcriptional regulators important for white-opaque switching: Wor1, Wor2, Wor3, Czf1, Efg1, and Ahr1. In this work, we describe the structure of the transcriptional network that specifies the white and opaque cell types and governs the ability to switch between them. In particular, we use a combination of genome-wide chromatin immunoprecipitation, gene expression profiling, and microfluidics-based DNA binding experiments to determine the direct and indirect regulatory interactions that form the switch network. The six regulators are arranged together in a complex, interlocking network with many seemingly redundant and overlapping connections. We propose that the structure (or topology) of this network is responsible for the epigenetic maintenance of the white and opaque states, the switching between them, and the specialized properties of each state.
doi:10.1111/mmi.12329
PMCID: PMC3888361  PMID: 23855748
White-opaque switching; Transcriptional Regulation; Transcription Networks; Transcriptional circuits; Candida albicans
6.  Profile Hidden Markov Models for the Detection of Viruses within Metagenomic Sequence Data 
PLoS ONE  2014;9(8):e105067.
Rapid, sensitive, and specific virus detection is an important component of clinical diagnostics. Massively parallel sequencing enables new diagnostic opportunities that complement traditional serological and PCR based techniques. While massively parallel sequencing promises the benefits of being more comprehensive and less biased than traditional approaches, it presents new analytical challenges, especially with respect to detection of pathogen sequences in metagenomic contexts. To a first approximation, the initial detection of viruses can be achieved simply through alignment of sequence reads or assembled contigs to a reference database of pathogen genomes with tools such as BLAST. However, recognition of highly divergent viral sequences is problematic, and may be further complicated by the inherently high mutation rates of some viral types, especially RNA viruses. In these cases, increased sensitivity may be achieved by leveraging position-specific information during the alignment process. Here, we constructed HMMER3-compatible profile hidden Markov models (profile HMMs) from all the virally annotated proteins in RefSeq in an automated fashion using a custom-built bioinformatic pipeline. We then tested the ability of these viral profile HMMs (“vFams”) to accurately classify sequences as viral or non-viral. Cross-validation experiments with full-length gene sequences showed that the vFams were able to recall 91% of left-out viral test sequences without erroneously classifying any non-viral sequences into viral protein clusters. Thorough reanalysis of previously published metagenomic datasets with a set of the best-performing vFams showed that they were more sensitive than BLAST for detecting sequences originating from more distant relatives of known viruses. To facilitate the use of the vFams for rapid detection of remote viral homologs in metagenomic data, we provide two sets of vFams, comprising more than 4,000 vFams each, in the HMMER3 format. We also provide the software necessary to build custom profile HMMs or update the vFams as more viruses are discovered (http://derisilab.ucsf.edu/software/vFam).
doi:10.1371/journal.pone.0105067
PMCID: PMC4139300  PMID: 25140992
7.  Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets 
Nucleic Acids Research  2013;41(12):5991-6004.
The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein’s DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2’s-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.
doi:10.1093/nar/gkt259
PMCID: PMC3695516  PMID: 23625967
8.  ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding 
mBio  2013;4(2):e00098-13.
ABSTRACT
Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIβ/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism.
IMPORTANCE
Multiple viruses use the same Golgi protein (ACBD3) to recruit the lipid kinase phosphatidylinositol 4-kinase class III beta (PI4KB) in order to replicate. We identify a new binding partner of ACBD3 in the evolutionarily conserved Rab GTPase-activating proteins (RabGAPs) TBC1D22A and -B. Interestingly, TBC1D22A directly competes with PI4KB for binding to the same location of ACBD3 by utilizing a similar binding domain. Different viruses are able to influence this interaction through distinct mechanisms to promote the association of PI4KB with ACBD3. This work informs our knowledge of both the physical interactions of the proteins that help maintain metazoan Golgi structure and how viruses subvert these evolutionarily conserved interactions for their own purposes.
doi:10.1128/mBio.00098-13
PMCID: PMC3622926  PMID: 23572552
9.  The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis 
PLoS Biology  2014;12(5):e1001861.
Here we demonstrate that RNAi can be used in molecular studies of the giant single-celled ciliate Stentor coeruleus, revealing morphogenetic functions of Mob1 and highlighting the potential of this classical model for studies of morphogenesis and regeneration.
Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.
Author Summary
Cells have the ability to develop complex morphologies, but the mechanisms that determine these varied shapes are not well understood. Cell shape determination can be challenging to study in multicellular organisms because it can be difficult to know whether shape changes are determined internally within an individual cell or externally, driven by input from neighboring cells or by both. The giant unicellular ciliate, Stentor coeruleus, provides an ideal single-cell model in which to study morphogenesis due to its large size and reproducible, complex patterning. Although Stentor was a popular experimental organism around 100 years ago, molecular tools were not subsequently developed to sustain its use as a model system today. Here we demonstrate that RNA interference (RNAi) “by feeding” is effective in Stentor and demonstrate its utility for studying morphogenesis and cell polarity patterning in this organism. We show that the conserved Mob1 kinase regulator protein is asymmetrically localized to the posterior end of Stentor and is positioned at the newly forming posterior pole during cell division, suggesting that it may have a role in morphogenesis. Using RNAi, we show that depletion of Mob1 results in Stentor cells with marked defects in morphogenesis. Our findings suggest that Stentor coeruleus can be a powerful model system studying morphogenesis and regeneration at the single-cell level and that Mob1 is a patterning protein required for its normal development and regeneration.
doi:10.1371/journal.pbio.1001861
PMCID: PMC4019465  PMID: 24823688
10.  Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease 
mBio  2012;3(4):e00180-12.
ABSTRACT
Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens.
IMPORTANCE
Inclusion body disease (IBD) is a common infectious disease of captive snakes. IBD is fatal and can cause the loss of entire animal collections. The cause of the disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal disease is significant since animals are the source of virtually every emerging infectious human disease. We searched for candidate causative agents in snakes diagnosed with IBD and found a group of novel viruses distantly related mainly to arenaviruses but also to filoviruses, both of which can cause fatal hemorrhagic fevers when transmitted from animals to humans. In addition to providing evidence that strongly suggests that these viruses cause snake IBD, this discovery reveals a new and unanticipated domain of virus biology and evolution.
doi:10.1128/mBio.00180-12
PMCID: PMC3419519  PMID: 22893382
11.  The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ 
Journal of Virology  2012;86(7):3605-3616.
The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIβ. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIβ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIβ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus. Point mutations in 3A that eliminate PI4KIIIβ association sensitized Aichi virus to PIK93, suggesting that disruption of the 3A/ACBD3/PI4KIIIβ complex may represent a novel target for therapeutic intervention that would be complementary to the inhibition of the kinase activity itself.
doi:10.1128/JVI.06778-11
PMCID: PMC3302542  PMID: 22258260
12.  Molecular Insights into the Biosynthesis of Guadinomine, a Type III Secretion System Inhibitor 
Journal of the American Chemical Society  2012;134(42):17797-17806.
Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC50 of 14 nM, guadinomine B is the most potent known inhibitor of the Type III Secretion System (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli, Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced, and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase – nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-ACP extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster, as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.
doi:10.1021/ja308622d
PMCID: PMC3483642  PMID: 23030602
13.  Global Identification of Peptidase Specificity by Multiplex Substrate Profiling 
Nature methods  2012;9(11):1095-1100.
A simple and rapid multiplex substrate profiling method has been developed to reveal the substrate specificity of any endo- or exo-peptidase using LC-MS/MS sequencing. A physicochemically diverse library of peptides was generated by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated prime and non-prime site information and substrate specificity matched or expanded upon previous substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C gene responsible for polypeptide processing and allowed Granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor ion abundance. Furthermore, the proteolytic secretions from a parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.
doi:10.1038/nmeth.2182
PMCID: PMC3707110  PMID: 23023596
14.  Synthesis and evaluation of 7-substituted 4-aminoquinoline analogs for antimalarial activity 
Journal of medicinal chemistry  2011;54(20):7084-7093.
We previously reported that substituted 4-aminoquinolines with a phenylether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amine’s alkyl substituents exhibited potent antimalarial activity against the multi-drug resistant strain P. falciparum W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogs, in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 and cytotoxicity mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1strain good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study.
doi:10.1021/jm200636z
PMCID: PMC3697074  PMID: 21910466
15.  PRICE: Software for the Targeted Assembly of Components of (Meta) Genomic Sequence Data 
G3: Genes|Genomes|Genetics  2013;3(5):865-880.
Low-cost DNA sequencing technologies have expanded the role for direct nucleic acid sequencing in the analysis of genomes, transcriptomes, and the metagenomes of whole ecosystems. Human and machine comprehension of such large datasets can be simplified via synthesis of sequence fragments into long, contiguous blocks of sequence (contigs), but most of the progress in the field of assembly has focused on genomes in isolation rather than metagenomes. Here, we present software for paired-read iterative contig extension (PRICE), a strategy for focused assembly of particular nucleic acid species using complex metagenomic data as input. We describe the assembly strategy implemented by PRICE and provide examples of its application to the sequence of particular genes, transcripts, and virus genomes from complex multicomponent datasets, including an assembly of the BCBL-1 strain of Kaposi’s sarcoma-associated herpesvirus. PRICE is open-source and available for free download (derisilab.ucsf.edu/software/price/ or sourceforge.net/projects/pricedenovo/).
doi:10.1534/g3.113.005967
PMCID: PMC3656733  PMID: 23550143
KSHV; de novo genome assembly; high-throughput DNA sequencing; metagenomics
16.  Bartonella quintana Deploys Host and Vector Temperature-Specific Transcriptomes 
PLoS ONE  2013;8(3):e58773.
The bacterial pathogen Bartonella quintana is passed between humans by body lice. B. quintana has adapted to both the human host and body louse vector niches, producing persistent infection with high titer bacterial loads in both the host (up to 105 colony-forming units [CFU]/ml) and vector (more than 108 CFU/ml). Using a novel custom microarray platform, we analyzed bacterial transcription at temperatures corresponding to the host (37°C) and vector (28°C), to probe for temperature-specific and growth phase-specific transcriptomes. We observed that transcription of 7% (93 genes) of the B. quintana genome is modified in response to change in growth phase, and that 5% (68 genes) of the genome is temperature-responsive. Among these transcriptional changes in response to temperature shift and growth phase was the induction of known B. quintana virulence genes and several previously unannotated genes. Hemin binding proteins, secretion systems, response regulators, and genes for invasion and cell attachment were prominent among the differentially-regulated B. quintana genes. This study represents the first analysis of global transcriptional responses by B. quintana. In addition, the in vivo experiments provide novel insight into the B. quintana transcriptional program within the body louse environment. These data and approaches will facilitate study of the adaptation mechanisms employed by Bartonella during the transition between human host and arthropod vector.
doi:10.1371/journal.pone.0058773
PMCID: PMC3595295  PMID: 23554923
17.  Identification and Manipulation of the Molecular Determinants Influencing Poliovirus Recombination 
PLoS Pathogens  2013;9(2):e1003164.
The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific “hotspots,” including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains.
Author Summary
Viral recombination is critical to understanding the evolution of viral groups and impacts vaccine design, but is poorly understood. In the poliovirus vaccine, recombination is one potential mode of failure where vaccine strains recombine to produce a pathogenic product. We combine gene synthesis and deep sequencing to generate a high-resolution recombination map of poliovirus, both as a model RNA virus and a continuing threat that has yet to be eradicated. This map shows that recombination is concentrated into hotspots and suggests that predictable and alterable motifs in the RNA sequence are associated with recombination frequency. We demonstrate the utility of these observations by re-designing a poliovirus strain to recombine more frequently than normal, facilitating future studies on the role of viral recombination during infection. This result suggests that a large-scale redesign of the entire poliovirus genome to dampen recombination may be feasible, with implications for producing safer and more stable live vaccines.
doi:10.1371/journal.ppat.1003164
PMCID: PMC3567174  PMID: 23408891
18.  Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution 
Genome Biology  2013;14(1):R10.
Background
Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.
Results
Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.
Conclusions
While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.
doi:10.1186/gb-2013-14-1-r10
PMCID: PMC4053949  PMID: 23363705
19.  Discovery of a Novel Polyomavirus in Acute Diarrheal Samples from Children 
PLoS ONE  2012;7(11):e49449.
Polyomaviruses are small circular DNA viruses associated with chronic infections and tumors in both human and animal hosts. Using an unbiased deep sequencing approach, we identified a novel, highly divergent polyomavirus, provisionally named MX polyomavirus (MXPyV), in stool samples from children. The ∼5.0 kB viral genome exhibits little overall homology (<46% amino acid identity) to known polyomaviruses, and, due to phylogenetic variation among its individual proteins, cannot be placed in any existing taxonomic group. PCR-based screening detected MXPyV in 28 of 834 (3.4%) fecal samples collected from California, Mexico, and Chile, and 1 of 136 (0.74%) of respiratory samples from Mexico, but not in blood or urine samples from immunocompromised patients. By quantitative PCR, the measured titers of MXPyV in human stool at 10% (weight/volume) were as high as 15,075 copies. No association was found between the presence of MXPyV and diarrhea, although girls were more likely to shed MXPyV in the stool than boys (p = 0.012). In one child, viral shedding was observed in two stools obtained 91 days apart, raising the possibility of chronic infection by MXPyV. A multiple sequence alignment revealed that MXPyV is a closely related variant of the recently reported MWPyV and HPyV10 polyomaviruses. Further studies will be important to determine the association, if any, of MXPyV with disease in humans.
doi:10.1371/journal.pone.0049449
PMCID: PMC3498111  PMID: 23166671
20.  Optimization of Propafenone Analogues as Anti-Malarial Leads 
Journal of medicinal chemistry  2011;54(21):7477-7485.
Propafenone, a class Ic antiarrythmic drug, inhibits growth of cultured Plasmodium falciparum. While the drug’s potency is significant, further development of propafenone as an antimalarial would require divorcing the antimalarial and cardiac activities as well as improving the pharmacokinetic profile of the drug. A small array of propafenone analogs was designed and synthesized to address the cardiac ion channel and PK liabilities. Testing of this array revealed potent inhibitors of the 3D7 (drug sensitive) and K1 (drug resistant) strains of P. falciparum that possessed significantly reduced ion channel effects and improved metabolic stability. Propafenone analogues are unusual among antimalarial leads in that they are more potent against the multi-drug resistant K1 strain of P. falciparum compared to the 3D7 strain.
doi:10.1021/jm2005546
PMCID: PMC3208124  PMID: 21955244
propafenone; malaria; microwave epoxide ring opening; hERG
21.  Splenic Red Pulp Macrophages Produce Type I Interferons as Early Sentinels of Malaria Infection but Are Dispensable for Control 
PLoS ONE  2012;7(10):e48126.
Type I interferons (T1IFNs) are among the earliest cytokines produced during infections due to their direct regulation by innate immune signaling pathways. Reports have suggested that T1IFNs are produced during malaria infection, but little is known about the in vivo cellular origins of T1IFNs or their role in protection. We have found that in addition to plasmacytoid dendritic cells, splenic red pulp macrophages (RPMs) can generate significant quantities of T1IFNs in response to P. chabaudi infection in a TLR9-, MYD88-, and IRF7-dependent manner. Furthermore, T1IFNs regulate expression of interferon-stimulated genes redundantly with Interferon-gamma (IFNG), resulting in redundancy for resistance to experimental malaria infection. Despite their role in sensing and promoting immune responses to infection, we observe that RPMs are dispensable for control of parasitemia. Our results reveal that RPMs are early sentinels of malaria infection, but that effector mechanisms previously attributed to RPMs are not essential for control.
doi:10.1371/journal.pone.0048126
PMCID: PMC3483282  PMID: 23144737
22.  Complete genome sequence of an astrovirus identified in a domestic rabbit (Oryctolagus cuniculus) with gastroenteritis 
Virology Journal  2012;9:216.
A colony of domestic rabbits in Tennessee, USA, experienced a high-mortality (~90%) outbreak of enterocolitis. The clinical characteristics were one to six days of lethargy, bloating, and diarrhea, followed by death. Heavy intestinal coccidial load was a consistent finding as was mucoid enteropathy with cecal impaction. Preliminary analysis by electron microscopy revealed the presence of virus-like particles in the stool of one of the affected rabbits. Analysis using the Virochip, a viral detection microarray, suggested the presence of an astrovirus, and follow-up PCR and sequence determination revealed a previously uncharacterized member of that family. Metagenomic sequencing enabled the recovery of the complete viral genome, which contains the characteristic attributes of astrovirus genomes. Attempts to propagate the virus in tissue culture have yet to succeed. Although astroviruses cause gastroenteric disease in other mammals, the pathogenicity of this virus and the relationship to this outbreak remains to be determined. This study therefore defines a viral species and a potential rabbit pathogen.
doi:10.1186/1743-422X-9-216
PMCID: PMC3502403  PMID: 22998755
Rabbit astrovirus; Rabbit virus; Virus discovery; Virochip; Gastroenteritis; Diarrhea virus; Mucoid enteropathy; Enterocolitis
23.  Retraction: Identification of a Novel Gammaretrovirus in Prostate Tumors of Patients Homozygous for R462Q RNASEL Variant 
PLoS Pathogens  2012;8(9):10.1371/annotation/7e2efc01-2e9b-4e9b-aef0-87ab0e4e4732.
doi:10.1371/annotation/7e2efc01-2e9b-4e9b-aef0-87ab0e4e4732
PMCID: PMC3445601  PMID: 23028303
24.  In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer 
PLoS ONE  2012;7(9):e44954.
XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.
doi:10.1371/journal.pone.0044954
PMCID: PMC3445615  PMID: 23028701
25.  Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray 
Genome Biology  2003;4(2):R9.
DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation of the Plasmodium falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development.
Background
The worldwide persistence of drug-resistant Plasmodium falciparum, the most lethal variety of human malaria, is a global health concern. The P. falciparum sequencing project has brought new opportunities for identifying molecular targets for antimalarial drug and vaccine development.
Results
We developed a software package, ArrayOligoSelector, to design an open reading frame (ORF)-specific DNA microarray using the publicly available P. falciparum genome sequence. Each gene was represented by one or more long 70 mer oligonucleotides selected on the basis of uniqueness within the genome, exclusion of low-complexity sequence, balanced base composition and proximity to the 3' end. A first-generation microarray representing approximately 6,000 ORFs of the P. falciparum genome was constructed. Array performance was evaluated through the use of control oligonucleotide sets with increasing levels of introduced mutations, as well as traditional northern blotting. Using this array, we extensively characterized the gene-expression profile of the intraerythrocytic trophozoite and schizont stages of P. falciparum. The results revealed extensive transcriptional regulation of genes specialized for processes specific to these two stages.
Conclusions
DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation and exploration of the P. falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development.
doi:10.1186/gb-2003-4-2-r9
PMCID: PMC151308  PMID: 12620119

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