scholarly journals Tilting the balance between RNA interference and replication eradicatesLeishmaniaRNA virus 1 and mitigates the inflammatory response

2016 ◽  
Vol 113 (43) ◽  
pp. 11998-12005 ◽  
Author(s):  
Erin A. Brettmann ◽  
Jahangheer S. Shaik ◽  
Haroun Zangger ◽  
Lon-Fye Lye ◽  
F. Matthew Kuhlmann ◽  
...  
Keyword(s):  
High Efficiency ◽  
Rna Virus ◽  
Rnai Pathway ◽  
Leishmania Braziliensis ◽  
Stem Loop ◽  
Mucocutaneous Leishmaniasis ◽  
Virus Retention ◽  
Dsrna Genome

ManyLeishmania(Viannia) parasites harbor the double-stranded RNA virusLeishmania RNA virus 1(LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in bothLeishmania braziliensisandLeishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lackscis- ortrans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For bothL. braziliensisandL. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3–dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence ofL. braziliensis, theLeishmaniaspecies responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of otherLeishmaniaviruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi–viral relationships in evolution: one of balance rather than elimination.

scholarly journals Molecular basis of reovirus virulence. Role of the M2 gene.

1980 ◽  
Vol 152 (4) ◽  
pp. 853-868 ◽  
Author(s):  
D H Rubin ◽  
B N Fields
Keyword(s):  
Virulence Gene ◽  
Systemic Infection ◽  
Genome Segment ◽  
Dsrna Segment ◽  
Newborn Mice ◽  
Serotype 1 ◽  
Dsrna Genome ◽  
Outer Capsid

The mammalian reoviruses (serotype 1, strain Lang and serotype 3, strain Dearing) differ in their sensitivity to digestion by chymotrypsin. We have found that the M2 double-stranded RNA (dsRNA) genome segment (encoding the micro1C outer capsid polypeptide) is responsible for this property. In addition to determining response to protease treatement in vitro, we have found that the M2 genome segment also determines the ability of these two viruses successfully to initiate local and systemic infection in newborn mice after peroral inoculation. Thus the M2 dsRNA segment defines a new virulence gene of the mammalian reoviruses.

scholarly journals Ribosomal pausing during translation of an RNA pseudoknot.

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase

2005 ◽  
Vol 79 (16) ◽  
pp. 10608-10618 ◽  
Author(s):  
Zivile Panaviene ◽  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Replication Proteins ◽  
Rna Sequences ◽  
Alternative Structures ◽  
Recognition Element ◽  
Rna Elements

ABSTRACT Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3′-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

scholarly journals Caenorhabditis elegans nuclear RNAi factor SET-32 deposits the transgenerational histone modification, H3K23me3

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lianna Schwartz-Orbach ◽  
Chenzhen Zhang ◽  
Simone Sidoli ◽  
Richa Amin ◽  
Diljeet Kaur ◽  
...  
Keyword(s):  
Chromatin Modification ◽  
Epigenetic Inheritance ◽  
Rnai Pathway ◽  
C Elegans ◽  
And Function ◽  
Nuclear Rnai ◽  
Factor Set

Nuclear RNAi provides a highly tractable system to study RNA-mediated chromatin changes and epigenetic inheritance. Recent studies have indicated that the regulation and function of nuclear RNAi-mediated heterochromatin are highly complex. Our knowledge of histone modifications and the corresponding histonemodifying enzymes involved in the system remains limited. In this study, we show that the heterochromatin mark, H3K23me3, is induced by nuclear RNAi at both exogenous and endogenous targets in C. elegans. In addition, dsRNA-induced H3K23me3 can persist for multiple generations after the dsRNA exposure has stopped. We demonstrate that the histone methyltransferase SET-32, methylates H3K23 in vitro. Both set-32 and the germline nuclear RNAi Argonaute, hrde-1, are required for nuclear RNAi-induced H3K23me3 in vivo. Our data poise H3K23me3 as an additional chromatin modification in the nuclear RNAi pathway and provides the field with a new target for uncovering the role of heterochromatin in transgenerational epigenetic silencing.

scholarly journals Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

1984 ◽  
Vol 4 (8) ◽  
pp. 1605-1617 ◽  
Author(s):  
G Gaines ◽  
G Attardi
Keyword(s):  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
High Efficiency ◽  
Rrna Synthesis ◽  
Isolated Mitochondria ◽  
Highly Efficient ◽  
Light Strand

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.

scholarly journals Leishmania RNA virus exacerbates Leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Renan V. H. de Carvalho ◽  
Djalma S. Lima-Junior ◽  
Marcus Vinícius G. da Silva ◽  
Marisa Dilucca ◽  
Tamara S. Rodrigues ◽  
...  
Keyword(s):  
Nlrp3 Inflammasome ◽  
Rna Virus ◽  
Severe Form ◽  
Inflammasome Activation ◽  
Type I ◽  
Mucocutaneous Leishmaniasis ◽  
Nlrp3 Inflammasome Activation ◽  
Parasite Survival ◽  
Important Virulence Factor

AbstractLeishmania RNA virus (LRV) is an important virulence factor associated with the development of mucocutaneous Leishmaniasis, a severe form of the disease. LRV-mediated disease exacerbation relies on TLR3 activation, but downstream mechanisms remain largely unexplored. Here, we combine human and mouse data to demonstrate that LRV triggers TLR3 and TRIF to induce type I IFN production, which induces autophagy. This process results in ATG5-mediated degradation of NLRP3 and ASC, thereby limiting NLRP3 inflammasome activation in macrophages. Consistent with the known restricting role of NLRP3 for Leishmania replication, the signaling pathway triggered by LRV results in increased parasite survival and disease progression. In support of this data, we find that lesions in patients infected with LRV+ Leishmania are associated with reduced inflammasome activation and the development of mucocutaneous disease. Our findings reveal the mechanisms triggered by LRV that contribute to the development of the debilitating mucocutaneous form of Leishmaniasis.

scholarly journals Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

2012 ◽  
Vol 80 (4) ◽  
pp. 1361-1372 ◽  
Author(s):  
Shivangi Agarwal ◽  
Shivani Agarwal ◽  
Preeti Pancholi ◽  
Vijay Pancholi
Keyword(s):  
High Efficiency ◽  
Regulatory System ◽  
Gene Encoding ◽  
Content Type ◽  
Knockout Mutants ◽  
Catalytically Active

ABSTRACTStreptococcus pneumoniaeexploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase ofS. pneumoniae(StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerlessphpPknockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknownin vitroandin vivoevidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpPmutants. In particular, StkP (threonine)-phosphorylated RR06 bound to thecbpApromoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

scholarly journals The role of Dicer-dependent RNA interference in regulating cross-species communication during fungus-fungus interactions

2021 ◽  
Author(s):  
Edoardo Piombo ◽  
Ramesh Raju Vetukuri ◽  
Anders Broberg ◽  
Pruthvi B Kalyandurg ◽  
Sandeep Kushwaha ◽  
...  
Keyword(s):  
Rna Interference ◽  
Pathogenic Fungus ◽  
Hydrolytic Enzymes ◽  
Plant Diseases ◽  
Parasitic Fungus ◽  
Transcriptional Gene Silencing ◽  
Clonostachys Rosea ◽  
Stem Loop

Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs . Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated . In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea . Moreover, the Δ dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum , and reduced production of 62 secondary metabolites (SM) including yellow‐coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea Δ dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea , and 11 was upregulated in the Δ dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B . cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δ dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology.

VIRUS–HOST INTERACTIONS, WITH EMPHASIS ON CERTAIN CYTOPATHIC PHENOMENA

1967 ◽  
Vol 45 (8) ◽  
pp. 1221-1234 ◽  
Author(s):  
H. W. J. Ragetli
Keyword(s):  
Electron Microscopy ◽  
Rna Virus ◽  
Hydrolytic Enzymes ◽  
Symptom Expression ◽  
Plant Interactions ◽  
Host Interactions ◽  
Different Types ◽  
Virus Strains

The apparent role played by each participant in virus–plant interactions, as suggested by symptom expression, is discussed. To explain the markedly different reactions of identical hosts to closely related virus strains, it is proposed on the basis of present concepts regarding protein synthesis and RNA-virus replication, that the viral genome, insofar as it does not code for coat protein and for enzyme(s) required for replication of viral ribonucleic acid (RNA), may be "translated" into protein acting as the true cytopathic agent.Information on the nature of certain viral-cytopathogenic effects in hypersensitive and susceptible hosts, mainly as revealed by electron microscopy, is presented. The dramatic and fatal intracellular disorganization shown by hypersensitive hosts and suggestive of cytolytic processes invited a search for the presence and possible role of organelles containing hydrolytic enzymes (lysosomes). The in vitro detection of particle-bound acid phosphatase in different types of organelles, confirmed by electron microscopy, may explain at least some aspect of the observed cellular degeneration.

scholarly journals Androgen metabolism via 17β-hydroxysteroid dehydrogenase type 3 in mammalian and non-mammalian vertebrates: comparison of the human and the zebrafish enzyme

2005 ◽  
Vol 35 (2) ◽  
pp. 305-316 ◽  
Author(s):  
R Mindnich ◽  
F Haller ◽  
F Halbach ◽  
G Moeller ◽  
M Hrabé de Angelis ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
High Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Male And Female ◽  
Type 3 ◽  
Identification And Characterization ◽  
Subcellular Level

Formation and inactivation of testosterone is performed by various members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The main player in testosterone formation is considered to be 17β-HSD type 3, which catalyzes the reduction of androstenedione to testosterone with high efficiency and is almost exclusively expressed in testis. So far, only the mammalian homologs have been characterized but nothing is known about the role of 17β-HSD type 3 in other vertebrates. In this study, we describe the identification and characterization of the zebrafish homolog. We found zebrafish 17β-HSD type 3 to be expressed in embryogenesis from sphere to 84 h post-fertilization. Expression was also detected in various tissues of both male and female adults, but displayed sexual dimorphism. Interestingly, expression was not highest in male testis but in male liver. In female adults, strongest expression was observed in ovaries. At the subcellular level, both human and zebrafish 17β-HSD type 3 localize to the endoplasmic reticulum. The zebrafish enzyme in vitro effectively catalyzed the conversion of androstenedione to testosterone by use of NADPH as cofactor. Among further tested androgens epiandrosterone and dehydroepiandrosterone were accepted as substrates and reduced at C-17 by the human and the zebrafish enzyme. Androsterone and androstanedione though, were only substrates of human 17β-HSD type 3, not the zebrafish enzyme. Furthermore, we found that both enzymes can reduce 11-ketoandrostenedione as well as 11β-hydroxyandrostenedione at C-17 to the respective testosterone forms. Our results suggest that 17β-HSD type 3 might play slightly different roles in zebrafish compared with human although testosterone itself is likely to have similar functions in both organisms.

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