Host DNA Sequences Research Articles

PATHOEXTRACT: A BIOINFORMATIC PIPELINE FOR QUALITY CONTROL AND HOST DNA REMOVAL IN PLASMODIUM FALCIPARUM NGS DATA

Malaria, caused by Plasmodium falciparum, is a significant global health burden, particularly in sub-Saharan Africa. Deep sequencing (NGS) of parasite genomes has revolutionized our understanding of its biology and the emergence of drug resistance. However, the presence of host human DNA and other microbial contaminants within patient samples can hinder accurate and efficient parasite genome analysis. To address this challenge, we have developed PathoExtract, a robust bioinformatics pipeline that integrates commonly used tools into a streamlined workflow. PathoExtract leverages Snakemake, a workflow management system, to provide a flexible and reproducible framework for data processing. The pipeline incorporates rigorous quality control steps to identify and remove low-quality reads and contaminants. Host DNA and microbial sequences are effectively filtered out using a combination of alignment-based and alignment-free methods, ensuring that only Plasmodium falciparum reads are retained for downstream analysis.The pipeline offers an intuitive graphical user interface, making it accessible to researchers with varying levels of bioinformatics expertise. This user-friendly interface simplifies the process of running the pipeline, even for those unfamiliar with command-line tools. The code and documentation for PathoExtract are freely available at: https://github.com/stanlasso/DREPAL-PATHOEXTRACT.

HPV DNA Integration at Actionable Cancer-Related Genes Loci in HPV-Associated Carcinomas.

In HPV-associated carcinomas, some examples of cancer-related genes altered by viral insertion and corresponding to potential therapeutic targets have been described, but no quantitative assessment of these events, including poorly recurrent targets, has been reported to date. To document these occurrences, we built and analyzed a database comprised of 1455 cases, including HPV genotypes and tumor localizations. Host DNA sequences targeted by viral integration were classified as "non-recurrent" (one single reported case; 838 loci), "weakly recurrent" (two reported cases; 82 loci), and highly recurrent (≥3 cases; 43 loci). Whereas the overall rate of cancer-related target genes was 3.3% in the Gencode database, this rate increased to 6.5% in "non-recurrent", 11.4% in "weakly recurrent", and 40.1% in "highly recurrent" genes targeted by integration (p = 4.9 × 10-4). This rate was also significantly higher in tumors associated with high-risk HPV16/18/45 than other genotypes. Among the genes targeted by HPV insertion, 30.2% corresponded to direct or indirect druggable targets, a rate rising to 50% in "highly recurrent" targets. Using data from the literature and the DepMap 23Q4 release database, we found that genes targeted by viral insertion could be new candidates potentially involved in HPV-associated oncogenesis. A more systematic characterization of HPV/host fusion DNA sequences in HPV-associated cancers should provide a better knowledge of HPV-driven carcinogenesis and favor the development of personalize patient treatments.

DNA Barcoding of Argyrops filamentosus (Perciformes: Sparidae) Based on Mitochondrial COI Gene and Histopathological Evaluation of Gills Infected by Monogeneans

Background: Sparidae is a fish family that belongs to the order Perciformes and is commonly known as sea breams and porgies. Many species are included in this family and divided into 71 genera that could be barcoded using mitochondrial DNA genes. These fish are vulnerable to different parasitic taxa that affect fish status. The present study is aimed to confirm the molecular status of Argyrops filamentosus fish via the mitochondrial cytochrome c oxidase (COI) gene and to study the pathological changes of gills infested by monogenean parasites. Methods: Thirty Argyrops filamentosus fish were collected from Jeddah (Saudi Arabia) and identified molecularly via the mtCOI gene. Also, gills were isolated and examined microscopically for presence of monogeneans. Histopathological impacts of monogeneans on fish gills were studied in comparison to the gills of non-infected fish. Result: The DNA of fish species was barcoded and showed highly stringent criteria with the previously Argyrops filamentosus sequence data. The obtained host DNA sequences were deposited in NCBI database under accession number OP975758.1. Examination of the investigated fish gills revealed the presence of three monogenean species Protolamellodiscus senilobatus Kritsky, Jiménez-Ruiz and Sey, 2000, Acleotrema maculatus Morsy, El-Fayoumi and Fahmy (2014) and Haliotrema susanae Soo, 2019. Monogenean parasites penetrated deeply with their haptor to the gill lamella and caused damage and degeneration of epithelial cells leading to the formation of a cup-shaped depression. Therefore, the mtDNA gene has the ability for host identification and heavy monogeneans infections lead to severe damage to fish gills.

​Identification of Gnathiid Isopods as Ectoparasites of the Brown-spotted Grouper Epinephelus chlorostigma from the Red Sea of Saudi Arabia

Background: Gnathiids are considered excellent ectoparasites widely distributed along the fish body with a biphasic life cycle including parasitic larval stages and free-living adults. This study focused on gnathiids infecting the brown-spotted grouper fish and confirming the identity of the host species. Methods: Twenty Epinephelus chlorostigma (Serranidae) were collected from the Red Sea Coast (especially off Jeddah, Saudi Arabia) and examined for gnathiid fish parasites. Species identification of the larvae was based on morphological criteria of Smit and Basson (2002). In contrast, the taxonomy of fish species was supported by amplification and sequencing of host DNA via the mitochondrial DNA genes (COI and 16S rRNA) using gnathiids’ blood meals. Result: Praniza larvae of a gnathiid isopod were collected from the gill chambers of the examined fish. The prevalence rate was 60% and mean intensity of the pranzia larvae was 16. The pranzia larva is described microscopically and distinguished by large body size (3.51-5.90 mm), the presence of 8th teeth in the mandible, maxilliped with 5-7th teeth on the first palp and the endite hooked, pleotelson with concave anterolateral margins and rami of uropods extend its apex. The recovered larvae could be identified as pranzia larvae of Gnathia pantherina Smit and Basson, 2002 due to the morphological resemblance. The host species identity was confirmed molecularly with COI and 16S rRNA genes and showed highly stringent criteria with the previously E. chlorostigma sequence data. The obtained host DNA sequences were deposited in NCBI database under accession numbers ON384530.1 for COI and ON384544.1 for 16S rRNA.

The Rice ILI2 Locus Is a Bidirectional Target of the African Xanthomonas oryzae pv. oryzae Major Transcription Activator-like Effector TalC but Does Not Contribute to Disease Susceptibility.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.

Diagnosis of lung squamous cell carcinoma based on metagenomic Next-Generation Sequencing

BackgroundThe clinical treatment of patients suspected of pulmonary infections often rely on empirical antibiotics. However, preliminary diagnoses were based on clinical manifestations and conventional microbiological tests, which could later be proved wrong. In this case, we presented a patient whose initial diagnosis was lung abscess, but antibiotic treatments had no effect, and metagenomic Next-Generation Sequencing (mNGS) indicated presence of neoplasm.Case presentationA 62-year-old female was diagnosed with lung abscess at three different health facilities. However, mNGS of bronchoalveolar lavage fluid did not support pulmonary infections. Rather, the copy number variation analysis using host DNA sequences suggested neoplasm. Using H&E staining and immunohistochemistry of lung biopsy, the patient was eventually diagnosed with lung squamous cell carcinoma.ConclusionsmNGS not only detects pathogens and helps diagnose infectious diseases, but also has potential in detecting neoplasm via host chromosomal copy number analysis. This might be beneficial for febrile patients with unknown or complex etiology, especially when infectious diseases were initially suspected but empirical antibiotic regimen failed.

Genetic architecture of host proteins involved in SARS-CoV-2 infection

Understanding the genetic architecture of host proteins interacting with SARS-CoV-2 or mediating the maladaptive host response to COVID-19 can help to identify new or repurpose existing drugs targeting those proteins. We present a genetic discovery study of 179 such host proteins among 10,708 individuals using an aptamer-based technique. We identify 220 host DNA sequence variants acting in cis (MAF 0.01-49.9%) and explaining 0.3-70.9% of the variance of 97 of these proteins, including 45 with no previously known protein quantitative trait loci (pQTL) and 38 encoding current drug targets. Systematic characterization of pQTLs across the phenome identified protein-drug-disease links and evidence that putative viral interaction partners such as MARK3 affect immune response. Our results accelerate the evaluation and prioritization of new drug development programmes and repurposing of trials to prevent, treat or reduce adverse outcomes. Rapid sharing and detailed interrogation of results is facilitated through an interactive webserver (https://omicscience.org/apps/covidpgwas/).

Abstract A31: Genome-wide association study (GWAS) of host DNA sequence variation and the gut microbiome in the Multiethnic Cohort

Abstract Patterns of microbiome diversity vary across human populations, and although variation is largely driven by diet and lifestyle, genetically encoded differences between hosts may be important in shaping the microbiome and health outcomes, including cancer. We report preliminary results from a GWAS of the gut microbiome in 6,217 individuals from the Multiethnic Cohort Study, including African Americans, Japanese Americans, Native Hawaiians, Latinos, and Whites. Genome-wide SNP data was based on existing data from a variety of Illumina Infinium arrays (500,000 to 2.5 million single nucleotide polymorphisms (SNPs); n=4,363) as well as genotyping 1,853 individuals using the Illumina MEGA EX array. SNP imputation was conducted using a cosmopolitan reference panel of all 1000 Genomes samples. The stool microbiome was assessed by paired-end sequencing (Illumina MiSeq) of the16S rRNA gene (V1-3). SNP-genera association tests were conducted using linear regression of covariate-adjusted bacterial genera abundance quintiles on SNP genotype. The chi-square statistics were adjusted by the genomic inflation factor. A threshold of p=5 × 10−8 was used to determine genome-wide statistical significance. The covariate-adjusted genera values were computed as the residuals of a logistic ordinal regression of genera abundance quintiles on variables expected to affect the microbiome (i.e., age, sex, genetic ancestry proportions, sample month, and sequencing batch). Initial results yielded 22 genome-wide significant associations across SNPs in 15 different human chromosomes and 11 bacterial genera. Notably, Fusobacteria was significantly associated with star-related lipid transfer domain (STARD3, chromosome 13q13; p=2.8 × 10−8), voltage-dependent calcium channel gamma 3 subunit (CACNG3, chromosome 16p12; p=3.1 × 10−8), organic anion transporter polypeptide (OATP, SCLO2B1, chromosome 11q13; p=2.8 × 10−8), and E-cadherin (CDHR3, chromosome 7q22, p=1.6 × 10−8). Some Fusobacterial species have been associated with increased risk of colon tumors. Coprobacillus was significantly associated with ubiquitin modifier activating enzyme 2 (UBA2, chromosome 19q13, p=4.1 × 10−9). The pathogen Slackia was significantly associated with variants in the zinc finger 850 gene (ZNF850, chromosome 19p13; p=4.3 × 10−8). These results suggest that host gene variants may be important in shaping the microbiome and may influence bacterial pathogen-associated cancer outcomes. Citation Format: Meredith A. Hullar, Johanna W. Lampe, Timothy Randolph, Keith R. Curtis, Unhee Lim, Lynne R. Wilkens, Loic Le Marchand, Bruce S. Kristal, Kris R. Monroe, Kechen Zhao, Daniel Stram, Iona Cheng. Genome-wide association study (GWAS) of host DNA sequence variation and the gut microbiome in the Multiethnic Cohort [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A31.

Host DNA integrity within blood meals of hematophagous larval gnathiid isopods (Crustacea, Isopoda, Gnathiidae)

BackgroundJuvenile gnathiid isopods are common ectoparasites of marine fishes. Each of the three juvenile stages briefly attach to a host to obtain a blood meal but spend most of their time living in the substrate, thus making it difficult to determine patterns of host exploitation. Sequencing of host blood meals from wild-caught specimens is a promising tool to determine host identity. Although established protocols for this approach exist, certain challenges must be overcome when samples are subjected to typical field conditions that may contribute to DNA degradation. The goal of this study was to address a key methodological issue associated with molecular-based host identification from free-living, blood-engorged gnathiid isopods—the degradation of host DNA within blood meals. Here we have assessed the length of time host DNA within gnathiid blood meals can remain viable for positive host identification.MethodsJuvenile gnathiids were allowed to feed on fish of known species and subsets were preserved at 4-h intervals over 24 h and then every 24 h up to 5 days post-feeding. Host DNA extracted from gnathiid blood meals was sequenced to validate the integrity of host DNA at each time interval. DNA was also extracted from blood meals of wild-fed gnathiids for comparison. Attempts were also made to extract host DNA from metamorphosed juveniles.ResultsUsing a cox1 universal fish primer set, known fish host DNA sequences were successfully identified for nearly 100% of third-stage juvenile gnathiid blood meals, digested for up to 5 days post-feeding. For second-stage juveniles, host identification was 100% successful when gnathiids were preserved within 24 h of collection. Fish hosts were positively identified for 69% of sequences from wild-fed gnathiid isopods. Of the 31% of sequences not receiving a ≥ 98 % match to a sequence in GenBank, 25 sequences were of possible invertebrate origin.ConclusionsTo our knowledge, this is the first study to examine the degradation rate of gnathiid isopod blood meals. Determining the rate at which gnathiids digest their blood meal is an important step in ensuring the successful host identification by DNA-based methods in large field studies.

Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors

BackgroundCommon bacterial blight is a devastating bacterial disease of common bean (Phaseolus vulgaris) caused by Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli. These phylogenetically distant strains are able to cause similar symptoms on common bean, suggesting that they have acquired common genetic determinants of adaptation to common bean. Transcription Activator-Like (TAL) effectors are bacterial type III effectors that are able to induce the expression of host genes to promote infection or resistance. Their capacity to bind to a specific host DNA sequence suggests that they are potential candidates for host adaption.ResultsTo study the diversity of tal genes from Xanthomonas strains responsible for common bacterial blight of bean, whole genome sequences of 17 strains representing the diversity of X. citri pv. fuscans and X. phaseoli pv. phaseoli were obtained by single molecule real time sequencing. Analysis of these genomes revealed the existence of four tal genes named tal23A, tal20F, tal18G and tal18H, respectively. While tal20F and tal18G were chromosomic, tal23A and tal18H were carried on plasmids and shared between phylogenetically distant strains, therefore suggesting recent horizontal transfers of these genes between X. citri pv. fuscans and X. phaseoli pv. phaseoli strains. Strikingly, tal23A was present in all strains studied, suggesting that it played an important role in adaptation to common bean. In silico predictions of TAL effectors targets in the common bean genome suggested that TAL effectors shared by X. citri pv. fuscans and X. phaseoli pv. phaseoli strains target the promoters of genes of similar functions. This could be a trace of convergent evolution among TAL effectors from different phylogenetic groups, and comforts the hypothesis that TAL effectors have been implied in the adaptation to common bean.ConclusionsAltogether, our results favour a model where plasmidic TAL effectors are able to contribute to host adaptation by being horizontally transferred between distant lineages.

Identification of Hepatozoon erhardovae Krampitz, 1964 from bank voles (Myodes glareolus) and fleas in Southern Hungary.

In order to investigate the prevalence and life cycle of apicomplexan parasites, small mammals were live-trapped with modified Sherman traps in Southern Hungary between 2010 and 2012. Altogether, 528 rodents (Apodemus flavicollis Melchior, 1834, Apodemus agrarius Pallas, 1771, Myodes glareolus Schreber, 1780, Microtus agrestis Linnaeus, 1761, Mus musculus Linnaeus, 1758 and Micromys minutus Pallas, 1771) were collected and four shrews (Sorex spp.) were by-catched. Captured animals belonging to non-protected species were euthanized, and spleen samples were preserved for histological and molecular analyses. During the examination of spleen smears, Hepatozoon parasites were observed in eight out of 48 bank voles (M. glareolus). DNA was isolated from altogether 221 spleen samples, and 18S rDNA was amplified using two different PCR protocols. The eight bank vole samples were positive with PCR, but none of the other M. glareolus spleen samples or any of the tissue samples from other species were found to be infected. Sequenced amplicons were very similar to Hepatozoon spp. detected in M. glareolus in Spain and Poland. Ectoparasites were collected from the small mammal carcasses and from the vegetation. Hepatozoon DNA was not found in the 181 ticks removed from the small mammals or in the 162 ticks collected with flagging, but was detected in all three flea species (4/43 Megabothris turbidus Rothschild, 1909, 3/10 Ctenophthalmus assimilis Taschenberg, 1880 and 7/78 Ctenophthalmus agyrtes Heller, 1896). Based on gamont morphology, vertebrate and arthropod host species and DNA sequences, the parasites in our study can be identified as Hepatozoon erhardovae.

The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel.

The emerging microbial source tracking (MST) methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes high throughput sequencing (HTS) to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU) analysis was then performed, from which sequences were retained for downstream quantitative polymerase chain reaction (qPCR) marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae, and other bacteria. Two assays (Av4143 and Av163) identified most of the avian fecal samples and demonstrated sensitivity values of 91 and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216) identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.

The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia

Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1–V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations.

연속적 차분 확장 기반 가역 DNA 워터마킹

Of recent interests on high capacity DNA storage, DNA watermarking for DNA copyright protection, and DNA steganography for DNA secret communication are augmented, the reversible DNA watermarking is much needed both to embed the watermark without changing the functionality of organism and to perfectly recover the host DNA sequence. In this paper, we address two ways of DE based reversible DNA watermarking using noncoding DNA sequence. The reversible DNA watermarking should consider the string structure of a DNA sequence, the organism functionality, the perfect recovery, and the high embedding capacity. We convert the string sequence of four characters in noncoding region to the decimal coded values and embed the watermark bit into coded values by two ways; DE based multiple bits embedding (DE-MBE) using pairs of neighbor coded values and consecutive DE-MBE (C-DE-MBE). Two ways process the comparison searching to prevent the false start codon that produces false coding region. Experimental results verified that our ways have more high embedding capacity than conventional methods and produce no false start codon and recover perfectly the host sequence without the reference sequence. Especially C-DE-MBE can embed more high two times than DE-MBE.

Information Hiding in Noncoding DNA for DNA Steganography

This paper presents an information hiding method for DNA steganography with which a massive amount of data can be hidden in a noncoding strand. Our method maps the encrypted data to the DNA sequence using a numerical mapping table, before concealing it in the noncoding sequence using a secret key comprising sector length and the random number generator's seed. Our encoding algorithm is sector-based and reference dependent. Using modular arithmetic, we created a unique binary-base translation for every sector. By conducting a simulation study, we showed that our method could preserve amino acid information, extract hidden data without reference to the host DNA sequence, and detect the position of mutation error. Experimental results verified that our method produced higher data capacity than conventional methods, with a bpn (bit-per-nucleotide) value that ranged from approximately 1-2, depending on the selected sector length. Additionally, our novel method detected the positions of mutation errors by the presence of a parity base in each sector.

Sa1901 Bacterial DNA and Methylated Host DNA Sequences in Plasma Samples of Healthy and Colorectal Cancer Patients: A Whole-Metagenome Analysis

Sa1901 Bacterial DNA and Methylated Host DNA Sequences in Plasma Samples of Healthy and Colorectal Cancer Patients: A Whole-Metagenome Analysis

Meta'omic Analytic Techniques for Studying the Intestinal Microbiome

Nucleotide sequencing has become increasingly common and affordable, and is now a vital tool for studies of the human microbiome. Comprehensive microbial community surveys such as MetaHit and the Human Microbiome Project have described the composition and molecular functional profile of the healthy (normal) intestinal microbiome. This knowledge will increase our ability to analyze host and microbial DNA (genome) and RNA (transcriptome) sequences. Bioinformatic and statistical tools then can be used to identify dysbioses that might cause disease, and potential treatments. Analyses that identify perturbations in specific molecules can leverage thousands of culture-based isolate genomes to contextualize culture-independent sequences, or may integrate sequence data with whole-community functional assays such as metaproteomic or metabolomic analyses. We review the state of available systems-level models for studies of the intestinal microbiome, along with analytic techniques and tools that can be used to determine its functional capabilities in healthy and unhealthy individuals.

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.

Viroids: How to infect a host and cause disease without encoding proteins

Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the satellite RNA, does not exclude alternative mechanisms.

Structural basis for sequence-specific recognition of DNA by TAL effectors.

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.