scholarly journals Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasiteLeishmania

2017 ◽  
Vol 115 (3) ◽  
pp. E506-E515 ◽  
Author(s):  
Danyil Grybchuk ◽  
Natalia S. Akopyants ◽  
Alexei Y. Kostygov ◽  
Aleksandras Konovalovas ◽  
Lon-Fye Lye ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Rna Polymerase ◽  
Human Disease ◽  
Rna Viruses ◽  
Biological Role ◽  
Viral Diversity ◽  
Rna Dependent Rna Polymerase ◽  
Human Parasite ◽  
Viral Lineage

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasiteLeishmania(CrithidiaandLeptomonas), as well as plant-infectingPhytomonas.Leptomonas pyrrhocoriswas a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable asLeptomonas seymouribearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed “Leishbunyavirus” (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed “Leishbunyaviridae.” Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirusLeishmaniavirus(LRV1/2), implying that it was acquired at about the same time theLeishmaniabecame able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.

scholarly journals Suppression of RNA-Dependent RNA Polymerase 6 Favors the Accumulation of Potato Spindle Tuber Viroid in Nicotiana Benthamiana

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 345 ◽  
Author(s):  
Charith Raj Adkar-Purushothama ◽  
Jean-Pierre Perreault
Keyword(s):  
Nicotiana Benthamiana ◽  
Small Rnas ◽  
Rna Viruses ◽  
Fold Increase ◽  
Potato Spindle Tuber Viroid ◽  
Rna Dependent Rna Polymerase ◽  
Viroid Infection ◽  
Genes Encoding ◽  
Plant Genes

To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant’s growth, as was illustrated by the plant’s increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant’s susceptibility to viroid infection.

scholarly journals An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

2013 ◽  
Vol 94 (9) ◽  
pp. 1961-1971 ◽  
Author(s):  
Cindy S. E. Tan ◽  
Jody M. Hobson-Peters ◽  
Martin J. Stoermer ◽  
David P. Fairlie ◽  
Alexander A. Khromykh ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Genetic Interaction ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Rna Dependent Rna Polymerase ◽  
West Nile

The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46–49 in the αA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the αA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically co-immunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the αA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the αA3 mutations had a significant effect on the MTase–POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase–POL interactions.

scholarly journals Immunogenetics of leishmanial and mycobacterial infections: the Belem Family Study

1997 ◽  
Vol 352 (1359) ◽  
pp. 1331-1345 ◽  
Author(s):  
J. M. Blackwell ◽  
G. F. Black ◽  
C. S. Peacock ◽  
E. N. Miller ◽  
D. Sibthorpe ◽  
...  
Keyword(s):  
Immune Response ◽  
Visceral Leishmaniasis ◽  
Candidate Gene ◽  
Mouse Chromosome ◽  
Human Disease ◽  
Disease Susceptibility ◽  
Allelic Association ◽  
Chromosome 11 ◽  
Mycobacterial Infections

In the 1970s and 1980s, analysis of recombinant inbred, congenic and recombinant haplotype mouse strains permitted us to effectively ‘scan’ the murine genome for genes controlling resistance and susceptibility to leishmanial infections. Five major regions of the genome were implicated in the control of infections caused by different Leishmania species which, because they show conserved synteny with regions of the human genome, immediately provides candidate gene regions for human disease susceptibility genes. A common intramacrophage niche for leishmanial and mycobacterial pathogens, and a similar spectrum of immune response and disease phenotypes, also led to the prediction that the same genes/candidate gene regions might be responsible for genetic susceptibility to mycobacterial infections such as leprosy and tuberculosis. Indeed, one of the murine genes ( Nramp1 ) was identified for its role in controlling a range of intramacrophage pathogens including leishmania, salmonella and mycobacterium infections. In recent studies, multicase family data on visceral leishmaniasis and the mycobacterial diseases, tuberculosis and leprosy, have been collected from north–eastern Brazil and analysed to determine the role of these candidate genes/regions in determining disease susceptibility. Complex segregation analysis provides evidence for one or two major genes controlling susceptibility to tuberculosis in this population. Family–based linkage analyses (combined segregation and linkage analysis; sib–pair analysis), which have the power to detect linkage between marker loci in candidate gene regions and the putative disease susceptibility genes over 10–;20 centimorgans, and transmission disequilibrium testing, which detects allelic associations over 1 centimorgan ( ca. 1 megabase), have been used to examine the role of four regions in determining disease susceptibility and/or immune response phenotype. Our results demonstrate: (i) the major histocompatibility complex (MHC: H–2 in mouse, HLA in man: mouse chromosome 17/human 6p; candidates class II and class III including TNFalpha/beta genes) shows both linkage to, and allelic association with, leprosy per se , but is only weakly associated with visceral leishmaniasis and shows neither linkage to nor allelic association with tuberculosis; (ii) no evidence for linkage between NRAMP1 , the positionally cloned candidate for the murine macrophage resistance gene Ity/Lsh/Bcg (mouse chromosome 1/human 2q35), and susceptibility to tuberculosis or visceral leishmaniasis could be demonstrated in this Brazilian population; (iii) the region of human chromosome 17q (candidates NOS2A , SCYA2–5 ) homologous with distal mouse chromosome 11, originally identified as carrying the Scl1 gene controlling healing versus nonhealing responses to Leishmania major , is linked to tuberculosis susceptibility; and (iv) the ‘T helper 2’ cytokine gene cluster (proximal murine chromosome 11/human 5q; candidates IL4, IL5, IL9, IRF1, CD14) controlling later phases of murine L. major infection, is not linked to human disease susceptibility for any of the three infections, but shows linkage to and highly significant allelic association with ability to mount an immune response to mycobacterial antigens. These studies demonstrate that the ‘mouse–to–man’ strategy, refined by our knowledge of the human immune response to infection, can lead to the identification of important candidate gene regions in man.

scholarly journals The Escherichia coli Regulator of Sigma 70 Protein, Rsd, Can Up-Regulate Some Stress-Dependent Promoters by Sequestering Sigma 70

2007 ◽  
Vol 189 (9) ◽  
pp. 3489-3495 ◽  
Author(s):  
Jennie E. Mitchell ◽  
Taku Oshima ◽  
Sarah E. Piper ◽  
Christine L. Webster ◽  
Lars F. Westblade ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Hybrid System ◽  
Transcriptome Analysis ◽  
Biological Role ◽  
Stress Dependent ◽  
Sigma 70 ◽  
Two Hybrid ◽  
Stimulation Of

ABSTRACT The Escherichia coli Rsd protein forms complexes with the RNA polymerase σ70 factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative σ38 factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with σ70. Our experiments support a model in which the role of Rsd is primarily to sequester σ70, thereby increasing the levels of RNA polymerase containing the alternative σ38 factor.

scholarly journals Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses

2020 ◽  
Author(s):  
Yuto Chiba ◽  
Takashi Yaguchi ◽  
Syun-ichi Urayama ◽  
Daisuke Hagiwara
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Current Knowledge ◽  
Single Gene ◽  
Rna Seq ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Viral Genomes ◽  
True Diversity ◽  
Fungal Viruses

AbstractBy identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42% of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp. By highlighting the limitations of conventional surveillance methods for RNA viruses, we showcase the potential of FLDS technology to broaden current knowledge about these viruses.Author SummaryThe development of RNA-seq technology has facilitated the discovery of RNA viruses in all types of biological samples. However, it is technically difficult to detect highly novel viruses using RNA-seq. We successfully reconstructed the genomes of multiple novel fungal RNA viruses by screening host fungi using a new technology, FLDS. Surprisingly, we identified two viral species whose RNA-dependent RNA polymerase (RdRp) proteins were separately encoded on different genome segments, overturning the commonly accepted view of the positional unity of RdRp proteins in viral genomes. This new perspective on divided RdRp proteins should hasten the discovery of viruses with unique RdRp structures that have been overlooked, and further advance current knowledge and understanding of the diversity and evolution of RNA viruses.

scholarly journals Late Male-Killing Viruses in Homona magnanima Identified as Osugoroshi Viruses, Novel Members of Partitiviridae

2021 ◽  
Vol 11 ◽  
Author(s):  
Ryosuke Fujita ◽  
Maki N. Inoue ◽  
Takumi Takamatsu ◽  
Hiroshi Arai ◽  
Mayu Nishino ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Rna Polymerase ◽  
Rna Viruses ◽  
Rna Virus ◽  
Horizontal Transmission ◽  
Rna Dependent Rna Polymerase ◽  
Double Stranded Rna ◽  
First Report ◽  
Male Killing ◽  
Male Specific

Late male-killing, a male-specific death after hatching, is a unique phenotype found in Homona magnanima, oriental tea tortrix. The male-killing agent was suspected to be an RNA virus, but details were unknown. We herein successfully isolated and identified the putative male-killing virus as Osugoroshi viruses (OGVs). The three RNA-dependent RNA polymerase genes detected were phylogenetically related to Partitiviridae, a group of segmented double-stranded RNA viruses. Purified dsRNA from a late male-killing strain of H. magnanima revealed 24 segments, in addition to the RdRps, with consensus terminal sequences. These segments included the previously found male-killing agents MK1068 (herein OGV-related RNA16) and MK1241 (OGV-related RNA7) RNAs. Ultramicroscopic observation of purified virions, which induced late male-killing in the progeny of injected moths, showed sizes typical of Partitiviridae. Mathematical modeling showed the importance of late male-killing in facilitating horizontal transmission of OGVs in an H. magnanima population. This study is the first report on the isolation of partiti-like virus from insects, and one thought to be associated with late male-killing, although the viral genomic contents and combinations in each virus are still unknown.

scholarly journals Senecavirus-Specific Recombination Assays Reveal the Intimate Link between Polymerase Fidelity and RNA Recombination

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Chen Li ◽  
Haiwei Wang ◽  
Jiabao Shi ◽  
Decheng Yang ◽  
Guohui Zhou ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Polymerase ◽  
Crucial Role ◽  
Virus Evolution ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Fidelity ◽  
Rdrp Region ◽  
Positive Strand Rna

ABSTRACTSenecavirus A (SVA) is a reemerging virus, and recent evidence has emphasized the importance of SVA recombinationin vivoon virus evolution. In this study, we report the development of an infectious cDNA clone for the SVA/HLJ/CHA/2016 strain. We used this strain to develop a reporter virus expressing enhanced green fluorescent protein (eGFP), which we then used to screen for a recombination-deficient SVA by an eGFP retention assay. Sequencing of the virus that retained the eGFP following passage allowed us to identify the nonsynonymous mutations (S460L alone and I212V-S460L in combination) in the RNA-dependent RNA polymerase (RdRp) region of the genome. We developed a Senecavirus-specific cell culture-based recombination assay, which we used to elucidate the role of RdRp in SVA recombination. Our results demonstrate that these two polymerase variants (S460L and I212/S460L) have reduced recombination capacity. These results indicate that the RdRp plays a central role in SVA replicative recombination. Notably, our results showed that the two recombination-deficient variants have higher replication fidelity than the wild type (WT) and display decreased ribavirin sensitivity compared to the WT. In addition, these two mutants exhibited significantly increased fitnessin vitrocompared to the WT. These results demonstrate that recombination and mutation rates are intimately linked. Our results have important implications for understanding the crucial role of the RdRp in virus recombination and fitness, especially in the molecular mechanisms of SVA evolution and pathogenicity.IMPORTANCERecent evidence has emphasized the importance of SVA recombination on virus evolutionin vivo. We describe the first assays to study Senecavirus A recombination. The results show that the RNA-dependent RNA polymerase plays a crucial role in recombination and that recombination can impact the fitness of SVA in cell culture. Further, SVA polymerase fidelity is closely related to recombination efficiency. The results provide key insights into the role of recombination in positive-strand RNA viruses.

Targeting Viral RNA-Dependent RNA Polymerases as an Antiviral Therapy

2021 ◽  
Vol 28 ◽  
Author(s):  
Daniel Miranda ◽  
David Jesse Sanchez
Keyword(s):  
Clinical Trials ◽  
Global Health ◽  
Rna Polymerase ◽  
Biomedical Research ◽  
Rna Viruses ◽  
Standard Of Care ◽  
Rna Dependent Rna Polymerase ◽  
Viral Pathogens ◽  
Relevant Topic ◽  
Target Rna

Abstract: Progressive globalization of our society brings not only worldwide integration, it increases and promotes our exposure to new viral pathogens with evident impacts on our global health. Especially with the emergence of SARS-CoV-2, our biomedical research infrastructure has never been more compelled to rapidly develop antiviral regimens that demonstrate improved efficacy against these pathogens. Here we showcase 3 poignant antivirals against the lucrative target, RNA-dependent RNA polymerase (RdRP) of RNA viruses – a timely and relevant topic given the present efforts against COVID-19. While effective drug designs against RdRP are important, their benefit and potential as a standard of care truly relies on them standing out in well-designed clinical trials.

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