scholarly journals A Primary Determinant of Cap-Independent Translation Is Located in the 3′-Proximal Region of the Tomato Bushy Stunt Virus Genome

1999 ◽  
Vol 73 (11) ◽  
pp. 8982-8988 ◽  
Author(s):  
Baodong Wu ◽  
K. Andrew White
Keyword(s):  
Rna Virus ◽  
Present Report ◽  
Tomato Bushy Stunt Virus ◽  
Translational Efficiency ◽  
Genome Replication ◽  
Appreciable Effect ◽  
Positive Strand Rna ◽  
Independent Translation

ABSTRACT Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus Tombusvirus. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5′ cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3′ terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845–5851, 1998). In the present report, we provide evidence that a 3′-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5′ cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3′ proximal element, designated as the 3′ cap-independent translational enhancer (3′CITE), that is required for proficient cap-independent translation; (iv) the 3′CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3′CITE activity; and (vi) no significant 3′CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3′CITE from other characterized plant viral 3′-proximal cap-independent translational enhancers. Additionally, because the 3′CITE overlaps withcis-acting replication signals, it could potentially participate in regulating the initiation of genome replication.

scholarly journals Entomophthovirus: An insect-derived iflavirus that infects a behavior manipulating fungal pathogen of dipterans

2018 ◽  
Author(s):  
Maxwell C. Coyle ◽  
Carolyn N. Elya ◽  
Michael Bronski ◽  
Michael B. Eisen
Keyword(s):  
Fungal Pathogen ◽  
Rna Virus ◽  
Delia Radicum ◽  
Sequencing Data ◽  
Entomophthora Muscae ◽  
Positive Strand Rna Virus ◽  
Close Relatives ◽  
Positive Strand Rna

AbstractWe discovered a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Entomophthovirus, is a capsid-forming, positive-strand RNA virus in the viral family iflaviridae, whose known members almost exclusively infect insects. We show that the virus RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and that virus particles are present in E. muscae. Two close relatives of the virus had been previously described as insect viruses based on the presence of viral genomes in transcriptomes assembled from RNA extracted from wild dipterans. By analyzing sequencing data from these earlier reports, we show that both dipteran samples were co-infected with E. muscae. We also find the virus in RNA sequencing data from samples of two other species of dipterans, Musca domestica and Delia radicum, known to be infected with E. muscae. These data establish that Entomophthovirus is widely, and seemingly obligately, associated with E. muscae. As other members of the iflaviridae cause behavioral changes in insects, we speculate on the possibility that Entomophthovirus plays a role in E. muscae involved host manipulation.

scholarly journals trans Regulation of Cap-Independent Translation by a Viral Subgenomic RNA

2006 ◽  
Vol 80 (20) ◽  
pp. 10045-10054 ◽  
Author(s):  
Ruizhong Shen ◽  
Aurélie M. Rakotondrafara ◽  
W. Allen Miller
Keyword(s):  
Barley Yellow Dwarf Virus ◽  
Viral Mrnas ◽  
Stem Loop ◽  
Subgenomic Rna ◽  
Yellow Dwarf Virus ◽  
Trans Regulation ◽  
Positive Strand Rna ◽  
Independent Translation

ABSTRACT Many positive-strand RNA viruses generate 3′-coterminal subgenomic mRNAs to allow translation of 5′-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3′ untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5′ UTR to mediate translation initiation at the 5′ end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5′ UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5′ end of the stem-loop that kisses the 3′ BTE. Thus, the gRNA and sgRNA1 have “tuned” their expression efficiencies via the site in the 5′ UTR to which the 3′ BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.

scholarly journals RNA Virus Fidelity Mutants: A Useful Tool for Evolutionary Biology or a Complex Challenge?

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 600 ◽  
Author(s):  
Tiffany Kautz ◽  
Naomi Forrester
Keyword(s):  
Evolutionary Biology ◽  
Rna Viruses ◽  
Rna Virus ◽  
Large Population ◽  
Genome Replication ◽  
Population Sizes ◽  
Host Infection ◽  
Definition Of

RNA viruses replicate with low fidelity due to the error-prone nature of the RNA-dependent RNA polymerase, which generates approximately one mutation per round of genome replication. Due to the large population sizes produced by RNA viruses during replication, this results in a cloud of closely related virus variants during host infection, of which small increases or decreases in replication fidelity have been shown to result in virus attenuation in vivo, but not typically in vitro. Since the discovery of the first RNA virus fidelity mutants during the mid-aughts, the field has exploded with the identification of over 50 virus fidelity mutants distributed amongst 7 RNA virus families. This review summarizes the current RNA virus fidelity mutant literature, with a focus upon the definition of a fidelity mutant as well as methods to confirm any mutational changes associated with the fidelity mutant. Due to the complexity of such a definition, in addition to reports of unstable virus fidelity phenotypes, the future translational utility of these mutants and applications for basic science are examined.

scholarly journals Mapping of sequences in the 5’ region and 3’ UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249928
Author(s):  
Dinesh Babu Paudel ◽  
Hélène Sanfaçon
Keyword(s):  
Translation Initiation ◽  
Rna Virus ◽  
Ringspot Virus ◽  
General Mechanism ◽  
Coding Region ◽  
Initiation Mechanism ◽  
Tomato Ringspot Virus ◽  
Positive Strand Rna ◽  
Independent Translation

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5’-viral genome-linked protein (VPg) and have a 3’ polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3’ untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5’ regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5’ region and 3’ UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5’ region and 3’ UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5’ proximal coding region of the RNA2 polyprotein and in the RNA2 3’ UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3’ UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3’ UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5’ region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773–972) of the 1547 nt long 3’ UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

scholarly journals Purinergic Modulation of Interleukin-1β Release from Microglial Cells Stimulated with Bacterial Endotoxin

1997 ◽  
Vol 185 (3) ◽  
pp. 579-582 ◽  
Author(s):  
Davide Ferrari ◽  
Paola Chiozzi ◽  
Simonetta Falzoni ◽  
Stefania Hanau ◽  
Francesco Di  Virgilio
Keyword(s):  
Plasma Membrane ◽  
P2x7 Receptor ◽  
Purinergic Receptor ◽  
Present Report ◽  
Physiological Role ◽  
Bacterial Endotoxin ◽  
Microglial Cells

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.

Tissue specificity of type I collagen gene expression is determined at both transcriptional and post-transcriptional levels

1984 ◽  
Vol 4 (9) ◽  
pp. 1843-1852
Author(s):  
R J Focht ◽  
S L Adams
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Type I Collagen ◽  
Translational Efficiency ◽  
Type I ◽  
Collagen Gene ◽  
Differentiated Cells ◽  
Collagen Gene Expression

We analyzed the control of type I collagen synthesis in four kinds of differentiated cells from chicken embryos which synthesize very different amounts of the protein. Tendon, skin, and smooth muscle cells were found to have identical amounts of type I collagen RNAs; however, the RNAs had inherently different translatabilities, which were observed both in vivo and in vitro. Chondrocytes also had substantial amounts of type I collagen RNAs, even though they directed no detectable synthesis of the protein either in vivo or in vitro. Type I collagen RNAs in chondrocytes display altered electrophoretic mobilities, suggesting that in these cells the reduction in translational efficiency may be mediated in part by changes in the RNA structure. These data indicate that control of type I collagen gene expression is a complex process which is exerted at both transcriptional and post-transcriptional levels.

scholarly journals Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

2004 ◽  
Vol 78 (14) ◽  
pp. 7619-7633 ◽  
Author(s):  
Guohua Zhang ◽  
Jiuchun Zhang ◽  
Anne E. Simon
Keyword(s):  
Solution Structure ◽  
Rna Virus ◽  
Structural Features ◽  
General Mechanism ◽  
Minus Strand ◽  
Turnip Crinkle Virus ◽  
Structural Elements ◽  
Transcription In Vitro

ABSTRACT Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3′-terminal bases. Deletion of satC 3′-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3′ end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3′-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3′ terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3′ end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3′-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus.

scholarly journals Modeling of SARS-CoV-2 Treatment Effects for Informed Drug Repurposing

2021 ◽  
Vol 12 ◽  
Author(s):  
Charlotte Kern ◽  
Verena Schöning ◽  
Carlos Chaccour ◽  
Felix Hammann
Keyword(s):  
Drug Repurposing ◽  
Antiviral Effect ◽  
Drug Candidate ◽  
Appreciable Effect ◽  
Viral Kinetics ◽  
Future Drug ◽  
Dosing Regimens ◽  
Clinical Trial Results

Several repurposed drugs are currently under investigation in the fight against coronavirus disease 2019 (COVID-19). Candidates are often selected solely by their effective concentrations in vitro, an approach that has largely not lived up to expectations in COVID-19. Cell lines used in in vitro experiments are not necessarily representative of lung tissue. Yet, even if the proposed mode of action is indeed true, viral dynamics in vivo, host response, and concentration-time profiles must also be considered. Here we address the latter issue and describe a model of human SARS-CoV-2 viral kinetics with acquired immune response to investigate the dynamic impact of timing and dosing regimens of hydroxychloroquine, lopinavir/ritonavir, ivermectin, artemisinin, and nitazoxanide. We observed greatest benefits when treatments were given immediately at the time of diagnosis. Even interventions with minor antiviral effect may reduce host exposure if timed correctly. Ivermectin seems to be at least partially effective: given on positivity, peak viral load dropped by 0.3–0.6 log units and exposure by 8.8–22.3%. The other drugs had little to no appreciable effect. Given how well previous clinical trial results for hydroxychloroquine and lopinavir/ritonavir are explained by the models presented here, similar strategies should be considered in future drug candidate prioritization efforts.

scholarly journals Regulation of Translational Efficiency by Disparate5′-UTRs of PPARγSplice Variants

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Shawn McClelland ◽  
Roopali Shrivastava ◽  
Jheem D. Medh
Keyword(s):  
Secondary Structure ◽  
Posttranscriptional Regulation ◽  
Protein A ◽  
Firefly Luciferase ◽  
Translational Efficiency ◽  
Protein Levels ◽  
Gene Assay ◽  
Firefly Luciferase Gene

The PPAR-γgene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the5′-untranslated region (UTR). The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the5′-UTR in regulating the efficiency with which the message is translated to protein. A coupledin vitrotranscription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. Anin vivoreporter gene assay using each5′-UTR upstream of the firefly luciferase gene showed that the5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the5′-UTR, and the length of the5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.

scholarly journals A Unique 5′ Translation Element Discovered in Triticum Mosaic Virus

2015 ◽  
Vol 89 (24) ◽  
pp. 12427-12440 ◽  
Author(s):  
Robyn Roberts ◽  
Jincan Zhang ◽  
Laura K. Mayberry ◽  
Satyanarayana Tatineni ◽  
Karen S. Browning ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Translation Initiation ◽  
Plant Virus ◽  
Plant Viruses ◽  
Leader Sequence ◽  
Content Type ◽  
Internal Translation ◽  
Positive Strand Rna ◽  
Independent Translation

ABSTRACTSeveral plant viruses encode elements at the 5′ end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5′ m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to thePotyviridaefamily of positive-strand RNA viruses. We demonstrate that the TriMV 5′ leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5′ leader to a 300-nucleotide sequence that drives cap-independent translation from the 5′ end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5′ untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5′ UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5′ end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation.IMPORTANCEMany members of thePotyviridaefamily rely on their 5′ end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5′ leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5′ UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5′ UTR is capable of driving cap-independent translationin vitroandin vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNAin vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

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