A plant-infecting subviral RNA associated with poleroviruses produces a subgenomic RNA which resists exonuclease XRN1 in vitro

ABSTRACT Tulane virus (TV) is a newly reported calicivirus that was isolated from stool samples of captive rhesus macaques from the Tulane National Primate Research Center (TNPRC). The virus has been cultivated successfully in LLC-MK2 rhesus monkey kidney cells. Its complete genomic sequence suggests that TV represents a new genus and is evolutionarily more closely related to Norovirus than to any other genus of Caliciviridae. In this study, we demonstrated that RNA transcripts made in vitro from the full-length genomic cDNA of TV were infectious upon transfection into permissive LLC-MK2 cells. The recombinant virus exhibited plaque morphologies and growth kinetics similar to those of the wild-type virus in this cell line. Capping was required for TV RNA infectivity. Although a subgenomic RNA has been detected in TV-transfected cells, a separate subgenomic RNA transcript was not required for the initial transfection to establish the replication. Transfection of truncated RNA lacking open reading frame 2 (ORF2) and ORF3 or TV-norovirus chimeric RNA resulted in abortive replication without the production of infectious progeny viruses, indicating that both ORFs are essential for the replication of TV. A heterologous insertion at the 5′ end of the genome also hampered viral replication, suggesting that an authentic 5′ end of the genome is critical for replication. The availability of the complete genomic sequence and the reverse genetics system described herein make TV a valuable model for studying calicivirus pathogenesis and replication.

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Cell Clones Selected from the Huh7 Human Hepatoma Cell Line Support Efficient Replication of a Subgenomic GB Virus B Replicon

Journal of Virology ◽

10.1128/jvi.76.15.7736-7746.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7736-7746 ◽

Cited By ~ 20

Author(s):

Amedeo De Tomassi ◽

Maura Pizzuti ◽

Rita Graziani ◽

Andrea Sbardellati ◽

Sergio Altamura ◽

...

Keyword(s):

Host Cell ◽

Cell Line ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Human Hepatoma ◽

Human Hepatoma Cell ◽

Subgenomic Rna ◽

Human Hepatoma Cell Line ◽

Huh7 Cells

ABSTRACT Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.

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Studies on the translation mechanism of subgenomic RNA of potato leafroll virus.

Acta Biochimica Polonica ◽

10.18388/abp.1997_4441 ◽

1997 ◽

Vol 44 (1) ◽

pp. 69-77 ◽

Cited By ~ 1

Author(s):

M Juszczuk ◽

W Zagórski-Ostoja ◽

D M Hulanicka

Keyword(s):

Stop Codon ◽

In Vitro Transcription ◽

Open Reading Frames ◽

Translation System ◽

Translation Efficiency ◽

Subgenomic Rna ◽

Expression Of Genes ◽

Initiation Of Translation ◽

Leafroll Virus

The expression of open reading frames located on the subgenomic RNA (sgRNA) has been studied in an in vitro transcription and translation system. The obtained results indicate: a) translation of sgRNA occurs according to the scanning model since the insertion of palindrome (delta G0 = -61 kcal/mol) prevents the initiation of translation; b) ORF6 is translated by suppression of the stop codon separating ORF4 from ORF6 and the presence of suppressor tRNA is necessary for the readthrough; c) the presence of leader sequence of sgRNA (212 nucleotides) decreases the translation efficiency of ORFs located downstream and it affects the ratio of products of ORF4 and ORF5; d) 3'UTR does not influence on an expression of genes located on the sgRNA.

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Ilarviruses Encode a Cucumovirus-Like 2b Gene That Is Absent in Other Genera within the Bromoviridae

Journal of Virology ◽

10.1128/jvi.72.8.6956-6959.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6956-6959 ◽

Cited By ~ 33

Author(s):

Hong-Wu Xin ◽

Liang-Hui Ji ◽

Simon W. Scott ◽

Robert H. Symons ◽

Shou-Wei Ding

Keyword(s):

Open Reading Frame ◽

Latent Virus ◽

Evolutionary Origin ◽

Protein Species ◽

Subgenomic Rna ◽

Reading Frame ◽

2B Protein ◽

Sequence Similarities

ABSTRACT We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3′ end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

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Evidence for the role of subgenomic RNA species in the production of Helenium virus S coat protein during in vitro translation

Virus Research ◽

10.1016/0168-1702(90)90080-u ◽

1990 ◽

Vol 17 (1) ◽

pp. 61-69 ◽

Cited By ~ 7

Author(s):

Gary Foster ◽

Peter Mills

Keyword(s):

Coat Protein ◽

In Vitro Translation ◽

Subgenomic Rna

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An RNA Pseudoknot Is Required for Production of Yellow Fever Virus Subgenomic RNA by the Host Nuclease XRN1

Journal of Virology ◽

10.1128/jvi.01047-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 11395-11406 ◽

Cited By ~ 119

Author(s):

Patrícia A. G. C. Silva ◽

Carina F. Pereira ◽

Tim J. Dalebout ◽

Willy J. M. Spaan ◽

Peter J. Bredenbeek

Keyword(s):

Yellow Fever ◽

Rna Structure ◽

Mammalian Cells ◽

Yellow Fever Virus ◽

Fever Virus ◽

Infected Mosquito ◽

Molecular Characteristics ◽

Subgenomic Rna ◽

Rna Pseudoknot

ABSTRACT Cells and mice infected with arthropod-borne flaviviruses produce a small subgenomic RNA that is colinear with the distal part of the viral 3′-untranslated region (UTR). This small subgenomic flavivirus RNA (sfRNA) results from the incomplete degradation of the viral genome by the host 5′-3′ exonuclease XRN1. Production of the sfRNA is important for the pathogenicity of the virus. This study not only presents a detailed description of the yellow fever virus (YFV) sfRNA but, more importantly, describes for the first time the molecular characteristics of the stalling site for XRN1 in the flavivirus genome. Similar to the case for West Nile virus, the YFV sfRNA was produced by XRN1. However, in contrast to the case for other arthropod-borne flaviviruses, not one but two sfRNAs were detected in YFV-infected mammalian cells. The smaller of these two sfRNAs was not observed in infected mosquito cells. The larger sfRNA could also be produced in vitro by incubation with purified XRN1. These two YFV sfRNAs formed a 5′-nested set. The 5′ ends of the YFV sfRNAs were found to be just upstream of the previously predicted RNA pseudoknot PSK3. RNA structure probing and mutagenesis studies provided strong evidence that this pseudoknot structure was formed and served as the molecular signal to stall XRN1. The sequence involved in PSK3 formation was cloned into the Sinrep5 expression vector and shown to direct the production of an sfRNA-like RNA. These results underscore the importance of the RNA pseudoknot in stalling XRN1 and also demonstrate that it is the sole viral requirement for sfRNA production.

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Similarities and Differences between the Subgenomic and Minus-Strand Promoters of an RNA Plant Virus

Journal of Virology ◽

10.1128/jvi.78.8.4048-4053.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4048-4053 ◽

Cited By ~ 12

Author(s):

René C. L. Olsthoorn ◽

P. C. Joost Haasnoot ◽

John F. Bol

Keyword(s):

Plant Virus ◽

Alfalfa Mosaic Virus ◽

Structural Features ◽

Minus Strand ◽

Promoter Regions ◽

Subgenomic Rna ◽

Live Virus ◽

The Family ◽

Subgenomic Promoter

ABSTRACT Promoter regions required for minus-strand and subgenomic RNA synthesis have been mapped for several plus-strand RNA viruses. In general, the two types of promoters do not share structural features even though they are recognized by the same viral polymerase. The minus-strand promoter of Alfalfa mosaic virus (AMV), a plant virus of the family Bromoviridae, consists of a triloop hairpin (hpE) which is attached to a 3′ tRNA-like structure (TLS). In contrast, the AMV subgenomic promoter consists of a single triloop hairpin that bears no sequence homology with hpE. Here we show that hpE, when detached from its TLS, can function as a subgenomic promoter in vitro and can replace the authentic subgenomic promoter in the live virus. Thus, the AMV subgenomic and minus-strand promoters are basically the same, but the minus-strand promoter is linked to a 3′ TLS to force the polymerase to initiate at the very 3′end.

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Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (−)-sense genomic RNA

Nature ◽

10.1038/313068a0 ◽

1985 ◽

Vol 313 (5997) ◽

pp. 68-70 ◽

Cited By ~ 178

Author(s):

W. A. Miller ◽

T. W. Dreher ◽

T. C. Hall

Keyword(s):

Mosaic Virus ◽

Brome Mosaic Virus ◽

Genomic Rna ◽

Subgenomic Rna ◽

Internal Initiation

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The Effects of Ethyl Lauroyl Arginine Hydrochloride (ELAH) in Nasal Spray Formula on SAR-Cov-2

10.21203/rs.3.rs-911449/v1 ◽

2021 ◽

Author(s):

Harshad R. Thacore ◽

Abdul Gaffar ◽

Seiyoung Yun ◽

Agnes L. Chenine ◽

Maria G. Ferrari ◽

...

Keyword(s):

Respiratory Diseases ◽

Control Group ◽

Hamster Model ◽

Subgenomic Rna ◽

Disease Symptoms ◽

In Vivo Assays ◽

Antiviral Effects ◽

Arginine Hydrochloride

Abstract SARS-CoV-2 and coronaviruses, enveloped RNA viruses, are major causes of acute human respiratory diseases. The aim of the study was to investigate the broad-spectrum antiviral effects of ethyl lauroyl arginine hydrochloride (ELAH) in in vitro and in vivo assays. Cell-based assays found that the pseudovirus VSV-SARS-CoV-2 was inhibited with an EC50 of 15 micrograms/ml, with complete inhibition achieved at 110 micrograms/ml. The effects were comparable to those observed with anti-SARS-CoV-2 antibody neutralization assays against VSV-SARS-CoV-2. Intranasal administration of the Wuhan strain of SARS-CoV-2 treated in vitro with ELAH inhibited the disease symptoms caused by the virus in a Syrian hamster model compared to that caused by the same dose of virus treated in vitro with medium alone. Subgenomic RNA and total RNA viral load were concomitantly reduced in the treated animals compared with the control group. In cell-based studies, pretreatment of susceptible cells with 1-10 micrograms/ml ELAH inhibited the attachment of the virus to the cells, as measured by cytopathic and high-resolution scanning electron microscopy (SEM) effects, suggesting that the primary mode of ELAH action was due to preventing the attachment of the virus to the cells. Collectively, the data suggest that ELAH could be a promising agent for the prevention of SARS infection through nasopharyngeal surfaces.

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Requirements for Brome Mosaic Virus Subgenomic RNA Synthesis In Vivo and Replicase-Core Promoter Interactions In Vitro

Journal of Virology ◽

10.1128/jvi.78.12.6091-6101.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6091-6101 ◽

Cited By ~ 19

Author(s):

K. Sivakumaran ◽

Seung-Kook Choi ◽

Masarapu Hema ◽

C. Cheng Kao

Keyword(s):

Mosaic Virus ◽

Rna Synthesis ◽

Core Promoter ◽

Brome Mosaic Virus ◽

Wild Type ◽

Subgenomic Rna ◽

The Core ◽

Rna Hairpin

ABSTRACT Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

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