The role of retroviral dUTPases in replication and virulence

Several retroviruses, including equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV) and feline immunodeficiency virus (FIV) encode dUTPase. The role of this enzyme in the replication of these viruses has been scrutinized, with particular emphasis on potential roles for dUTPase in virulence and viral mutation rate. Overall, the results of these studies have indicated a central role for dUTPase in facilitating productive viral replication in non-dividing cells. The requirement for dUTPase in EIAV, which replicates exclusively in macrophages, may be the most stringent. Studies of dUTPase mutants of virulent EIAV clones suggest that the enzyme is a major determinant of virulence. In contrast, FIV readily replicates in dividing cell populations such as CD4+ and CD8+ T cells, and B cells as well as in non-dividing macrophages. Thus, the virus burden and disease sequelae are lowered in cats infected with a dUTPase-minus FIV relative to cats infected with wild type FIV, but not totally abrogated. Growth in macrophages is attenuated with the DU-minus FIV with evidence of a 5 to 8-fold increase in G-->A transition mutations in viral integrants present in macrophages. These findings are consistent with an increase in uracil misincorporation in the absence of dUTPase, resulting in transition mutations that cripple the virus. Effects on virus replication and disease production have also been noted for dUTPase-deleted CEAV and visna virus. While HIV and SIV do not encode dUTPase some reports suggest that other viral and host cell factors may substitute for its activity. Betaretroviruses also encode dUTPase and while several of these cause significant disease, the role of dUTPase in their replication and pathogenesis is currently unknown.     

A resampling strategy for studying robustness in virus detection pipelines

Next-generation sequencing is regularly used to identify viral sequences in DNA or RNA samples of infected hosts. A major step of most pipelines for virus detection is to map sequence reads against known virus genomes. Due to small differences between the sequences of related viruses, and due to several biological or technical errors, mapping underlies uncertainties. As a consequence, the resulting list of detected viruses can lack robustness.A new approach for generating artificial sequencing reads together with a strategy of resampling from the original findings is proposed that can help to assess the robustness of the originally identified list of viruses. From the original mapping result in form of a SAM file, a set of statistical distributions are derived. These are used in the resampling pipeline to generate new artificial reads which are again mapped versus the reference genomes. By summarizing the resampling procedure, the analyst receives information about whether the presence of a particular virus in the sample gains or losses evidence, and thus about the robustness of the original mapping list but also that of individual viruses in this list. To judge robustness, several indicators are derived from the resampling procedure such as the correlation between original and resampling read counts, or the statistical detection of outliers in the differences of read counts. Additionally, graphical illustrations of read count shifts via Sankey diagrams are provided.To demonstrate the use of the new approach, the resampling approach is applied to three real-world data samples, one of them with laboratory-confirmed Influenza sequences, and to artificially generated data where virus sequences have been spiked into the sequencing data of a host. By applying the resampling pipeline, several viruses drop from the original list while new viruses emerge, showing robustness of those viruses that remain in the list.The evaluation of the new approach shows that the resampling approach is helpful to analyze the viral content of a biological sample, to rate the robustness of original findings and to better show the overall distribution of findings. The method is also applicable to other virus detection pipelines based on read mapping.     

Mass spectrometry based proteomic studies on viruses and hosts--a review

In terms of proteomic research in the 21st century, the realm of virology is still regarded as an enormous challenge mainly brought by three aspects, namely, studying on the complex proteome of the virus with unexpected variations, developing more accurate analytical techniques as well as understanding viral pathogenesis and virus–host interaction dynamics. Progresses in these areas will be helpful to vaccine design and antiviral drugs discovery. Mass spectrometry based proteomics have shown exceptional display of capabilities, not only precisely identifying viral and cellular proteins that are functionally, structurally, and dynamically changed upon virus infection, but also enabling us to detect important pathway proteins. In addition, many isolation and purification techniques and quantitative strategies in conjunction with MS can significantly improve the sensitivity of mass spectrometry for detecting low-abundant proteins, replenishing the stock of virus proteome and enlarging the protein–protein interaction maps. Nevertheless, only a small proportion of the infectious viruses in both of animal and plant have been studied using this approach. As more virus and host genomes are being sequenced, MS-based proteomics is becoming an indispensable tool for virology. In this paper, we provide a brief review of the current technologies and their applications in studying selected viruses and hosts.     

Analysis of genome sequences for genes of cyanophycin metabolism: identifying putative cyanophycin metabolizing prokaryotes

CGP, a copolymer of aspartate and arginine, serves as a storage compound for nitrogen, carbon and energy in many cyanobacteria. Analysis of available genome sequences from prokaryotes identified ORFs putatively encoding proteins of high similarity to known cyanophycin synthetases and cyanophycinases from cyanobacteria in various strains of bacteria belonging to different phylogenetic taxa and not closely related to cyanobacteria. Genes of CGP metabolism occur in a wide range of bacteria exhibiting diverse metabolic capabilities, including aerobic and anaerobic respiration, fermentation, phototrophy and chemolithoautotrophy. This study identified different groups of cyanophycin synthetases and cyanophycinases, respectively, and proposes a collective terminology for the putative genes and enzymes of cyanophycin metabolism. Among 570 different microbial strains, whose genomes have been partially or completely sequenced and are publicly accessible, we identified 44 prokaryotes which possess a cyanophycin synthetase and are putatively able to synthesize CGP. From these, 31 prokaryotes harbor also a cyanophycinase enabling them to degrade CGP to dipeptides. From the latter, 24 strains possess in addition a dipeptidase necessary to hydrolyze beta-Asp-Arg dipeptides, thereby enabling them to completely utilize CGP. Therefore, CGP seems to have a much wider distribution among prokaryotes than previously recognized. Genes putatively encoding cyanophycin synthetase homologues were not identified in the genomes of Eukarya and Archaea and are therefore obviously only occurring in Eubacteria. In addition, the outcome of this detailed in silico analysis proposes to distinguish 10 different groups of cyanophycin synthetases.     

The herpesvirus encoded dUTPase as a potential chemotherapeutic target

The human herpesviruses are a well characterized group of viruses that are responsible for a wide spectrum of human diseases. Included in this group of pathogens are the alphaherpesviruses (herpes simplex types 1 and 2 and varicella-zoster virus), the betaherpesviruses (cytomegalovirus, human herpesvirus types 6 and 7) and the gammaherpesviruses (Epstein-Barr virus and human herpesvirus 8). An important feature of these viruses is that they cause latent infections that can be reactivated to cause disease. The herpesviruses encode for a large number of structural and non-structural proteins, and several of the non-structural proteins, such as thymidine kinase, DNA polymerase, and ribonucleotide reductase, have been utilized as targets for the development of anti-herpesvirus agents. Another herpesvirus encoded enzyme that has received little attention as a potential target for the development of specific anti-herpesvirus agents is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Furthermore, little is known concerning the role of the herpesviruses' encoded dUTPases in virus replication and in modulating the chemotherapeutic efficiency of other anti-herpes agents. Because of recent advances in molecular virology and biochemistry, it is now possible to rationally develop "designer" drugs based upon the structural/functional interaction of the drug with a specific viral protein. The purpose of this review is to describe previous studies demonstrating the potential use of the herpesvirus encoded dUTPase as a drug target, to describe problems associated with using the dUTPase as a target and to discuss new approaches that can be used.     

Genome-wide high-throughput mining of natural-product biosynthetic gene clusters by phage display

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.     

The role of dUTPase and uracil-DNA repair in cancer chemotherapy

Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the molecular mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion observed following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metabolism as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metabolism. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clinical studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer.     

Lessons from viral pandemics

Viruses are ubiquitous in every habitat and are the most abundant species on our planet. Most ancient are RNA-containing viruses. They may have contributed to the origin of life, since naked RNA-only viruses, the viroids, lack genetic information. They fulfil several criteria of life. Could such structures have contributed to life on exoplanets somewhere in the Universe? Viruses are innovative, can integrate into genomes thereby supplying novel information and immunity to the host, protecting against superinfection by exogenous viruses. They are the drivers of evolution. As such they contributed e. g. to the placenta in mammals. The human chromosomes consist of about 50 % viral elements. Sub-viral structures such as transposable elements can contribute to genetics beyond Darwinian mutations. Bacterial viruses, the phages, can help to recycle nutrients in the oceans and our intestines and can destroy multi-drug resistant bacteria as phage-therapy. They may be a way out, if antibiotics fail. Antibiotic resistance my soon become critical. Phage therapy is a hundred years old, but has been neglected. Novel technologies may be able to reactivate this technology.     

Innate immune sensing of coronavirus and viral evasion strategies

The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces interferons (IFNs) and cytokines that perform antiviral functions to eliminate invading viruses. Coronaviruses are single-stranded, positive-sense RNA viruses that have a broad range of animal hosts. Coronaviruses have evolved multiple means to evade host antiviral immune responses. Successful immune evasion by coronaviruses may enable the viruses to adapt to multiple species of host organisms. Coronavirus transmission from zoonotic hosts to humans has caused serious illnesses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease-2019 (COVID-19), resulting in global health and economic crises. In this review, we summarize the current knowledge of the mechanisms underlying host sensing of and innate immune responses against coronavirus invasion, as well as host immune evasion strategies of coronaviruses.Subject terms: Infection, Pattern recognition receptors, Immune evasion     

Molecular characterization of pigeon torque teno virus (PTTV) in Jiangsu province

The torque teno virus (TTV) is a recently discovered DNA virus that has been detected in many different hosts, including humans, livestock and poultry. To date, there is no report of pigeon TTV (PTTV) from anywhere in the world. To investigate the distribution of PTTV in pigeons from the eastern Chinese province of Jiangsu and characterize their genomes, we employed PCR to detect PTTV in 144 samples collected from 6 pigeon plants in Jiangsu province, amplify complete genomes from representative samples and analyze genetic characteristics using bioinformatics. The results demonstrated that 71.5% (103/144) of samples were PTTV positive. The rate of sequence homology among the six PTTV complete genomes obtained from Jiangsu province ranged from 99.7% to 100%. Phylogenetic analysis suggested that PTTV genomes had a high degree of genetic similarity and were similar to chicken anemia virus that also had poultry as a host. Although with the same host, PTTV shared distant relationship with PiCV in both complete genome, Rep and Cap genes. The results of this study provided evidence that PTTV could be detected in Chinese pigeons at a high level, the evolutionary process of complete genome, Rep and Cap genes of Anelloviridae family had obvious divergence.     

Rapid determination of genomic DNA length for new bacteriophages

dsDNA viruses with long genomes (>200 kb) are expected to be a major source of novel genes. To rapidly characterize the genomes of newly isolated dsDNA bacteriophages, we develop here a procedure for the PFGE of intact long DNA genomes from bacteriophage particles in unfractionated, infected cell lysates of either liquid or gelled cultures. The DNA used for PFGE is suitable for sequencing after extraction with phenol. The PFGE is tuned to the range of expected DNA lengths. This procedure bypasses the isolation of bacteriophage particles and is useful for PFGE analysis of DNA from dissected zones of bacteriophage plaques.     

Characterization of the secondary metabolite biosynthetic gene clusters in archaea

Secondary metabolites are a range of bioactive compounds yielded by bacteria, fungi and plants, etc. The published archaea genomic data provide the opportunity for efficient identification of secondary metabolite biosynthetic gene clusters (BGCs) by genome mining. However, the study of secondary metabolites in archaea is still rare. By using the antiSMASH, we found two main putative secondary metabolite BGCs, bacteriocin and terpene in 203 Archaea genomes. Compared with the genomes of Euryarchaeota that usually lives in less complexity of environment, the genomes of Crenarchaeota usually contained more abundant bacteriocin. In these archaea genomes, we also found the positive correlation between the abundance of bacteriocin and the abundance of CRISPR spacer, suggesting the bacteriocin might be a crucial component of the innate immune system that defense the microbe living in the common environment. The structure analysis of the bacteriocin gene clusters gave a clue that the assisted genes located at the edge of clusters evolved faster than the core biosynthetic genes. To the best of our knowledge, we are the first to systematically explore the distribution of secondary metabolites in archaea, and the investigation of the relationship between BGC and CRISPR spacer expands our understanding of the evolutionary dynamic of these functional molecules.     

Glycine rich P-loop motif in deoxyuridine pyrophosphatase

Deoxyuridine pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester bond of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for dTTP synthesis. Similar to other nucleotide binding proteins, dUTPase also consists of a sequence motif rich in glycine residues known as P-loop motif. The P-loop motif of the nucleotide binding proteins are involved in substrate binding, catalysis, recognition and regulation of activity. In dUTPase the function of the P-loop motif is not well understood. One of the main reasons for this limited information is the lack of the three-dimensional structure of a dUTPase enzyme with an ordered Gly-rich P-loop motif with a bound substrate and Mg(2+) ion. This review presents an insight into the role of Gly-rich P-loop motif in the function of dUTPase as revealed from the crystal structure. The analysis reveals the Gly-rich P-loop motif of dUTPase to be the longest in terms of its amino-acid composition as compared to other nucleotide binding proteins and exhibit a high-degree of sequence conservation among spectrum of species. The enzyme utilizes adaptive recognition to bind to the phosphate groups of the nucleotide. In particular, the alpha-beta phosphodiester bond adopts an unfavorable eclipsed conformation in the presence of the Gly-rich P-loop motif. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.     

Guanosine distribution and oxidation resistance in eight eukaryotic genomes

Reactive oxygen species that attack DNA are continuously generated in living cells. Both the guanosine (G) mole fraction and its distribution should affect the stability of genomes and their parts to oxidation. At a lesser G content, genomes should be more oxidation resistant or "ennobled". Oxidant scavenging by G's in nonessential parts of introns and intergenic domains should decrease G oxidation in the essential exons. To determine whether genomes are indeed ennobled and whether oxidant-scavenging domains exist in genomes, the relative rates of guanosine oxidation in average exons, introns, and intergenic domains were estimated. Comparison among genomes indicated that average exons are ennobled in the genomes of Caenorhabditis (worm), Arabidopsis (plant), Saccharomyces (yeast), Schizosaccharomyces (yeast), and Plasmodium (malaria parasite), and that average introns and intergenic domains are ennobled in these genomes and in the genome of Drosophila (fly). The exon oxidation rates estimated for these genomes were less than the rate for the hypothetical "standard" genome, with a 0.25 mole fraction of uniformly distributed G. For Plasmodium the rate was half of that estimated for the standard genome. Average exons were not ennobled in the human or fly genomes; their G distributions were comparable to that in the standard genome. Instead, their exons were situated between introns and intergenic domains that could protect them by oxidant scavenging, the G's of their introns and intergenic domains outnumbering those of their exons 50-fold in humans and 4-fold in flies. The G distribution in the Encephalitozoon (parasite) genome was not protective relative to that of the standard genome.     

Viruses More Friends than Foes

Viruses are normally defined as pathogens and have a bad reputation because of pandemics such as Influenza, HIV/AIDS, Ebola, and SARS. Most viruses are, however, not enemies or killers but play important roles in the origin, development and maintenance of life of all species on our planet. This is new information we learnt by new technologies such as sequencing. Viruses are the most successful species on Earth, they are ubiquitous, in the oceans, in our environment, in animals, plants, bacteria, up in the air, perhaps even in the universe, within our body and even as part of our genomes. They influence our health, our well‐being, mental properties, our gut microbiota including obesity, and may help to cope with multi‐drug‐resistant bacteria. There the phages, viruses of bacteria, raise hopes. Viruses built our immunity: viruses protect against viruses. We do not have to lay eggs – thanks to viruses! They are the drivers of evolution and adaptation to environmental changes, also e. g. in plankton. The success story of viruses started about 3.5 billion years ago when life began. Newly discovered giant viruses are almost bacteria in their composition, suggesting that the borderline between dead matter and life is continuous. There are many open questions – how did life begin, is there life on exoplanets, how to find it? Are virus‐like elements, viroids, important for the origin of life? Will viruses eliminate mankind [1]?     

Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria

Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.     

Design and utility of oligonucleotide gene probes for fungal polyketide synthases

BACKGROUND: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological ('combinatorial') synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi. RESULTS: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as beta-ketoacylsynthases (KS), beta-ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments from as-yet undiscovered PKS gene clusters. The fragments could be used in blotting experiments with either homologous or heterologous fungal genomic DNA. CONCLUSIONS: A number of sequences are presented which have high utility for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters.