scholarly journals Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

2020 ◽  
Author(s):  
RM Langsjoen ◽  
AE Muruato ◽  
SR Kunkel ◽  
E Jaworski ◽  
A Routh
Keyword(s):  
Tissue Culture ◽  
De Novo ◽  
Chronic Arthritis ◽  
Early Passage ◽  
Viral Genomes ◽  
Large Deletions ◽  
Defective Rnas ◽  
Defective Rna ◽  
Intracellular Virus

ABSTRACTAlphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and encapsidated compartments. Importantly, these trends were likewise observed in a murine model of CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.IMPORTANCEOur understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between capsid/E3 and E1/3’UTR regions and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally may exist as subgenome-derived transcripts.

scholarly journals Differential Alphavirus Defective RNA Diversity between Intracellular and Extracellular Compartments Is Driven by Subgenomic Recombination Events

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
R. M. Langsjoen ◽  
A. E. Muruato ◽  
S. R. Kunkel ◽  
E. Jaworski ◽  
A. Routh
Keyword(s):  
Tissue Culture ◽  
De Novo ◽  
Chronic Arthritis ◽  
Early Passage ◽  
Viral Rnas ◽  
Viral Genomes ◽  
Large Deletions ◽  
Defective Rnas ◽  
Intracellular Virus

ABSTRACT Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template. IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3′ untranslated region (UTR) and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.

scholarly journals In Vitro- and In Vivo-Generated Defective RNAs of Satellite Panicum Mosaic Virus Define cis-Acting RNA Elements Required for Replication and Movement

2000 ◽  
Vol 74 (5) ◽  
pp. 2247-2254 ◽  
Author(s):  
Wenping Qiu ◽  
Scholthof G. Karen-Beth
Keyword(s):  
Mosaic Virus ◽  
De Novo ◽  
Stop Codon ◽  
Dependent Manner ◽  
Defective Rnas ◽  
Systemic Spread ◽  
Rna Elements ◽  
Rna Domains ◽  
Rna Accumulation

ABSTRACT Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3′ untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5′ UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5′ UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential “trigger” for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

scholarly journals Deep Sequencing of Recombinant Virus Populations in Transgenic and Nontransgenic Plants Infected with Cucumber mosaic virus

2013 ◽  
Vol 26 (7) ◽  
pp. 801-811 ◽  
Author(s):  
Marco Morroni ◽  
Mireille Jacquemond ◽  
Mark Tepfer
Keyword(s):  
Transgenic Plants ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
De Novo ◽  
Recombinant Viruses ◽  
Viral Genomes ◽  
Large Deletions ◽  
Cp Gene ◽  
Viral Sequences ◽  
Virus Variability

Recombination is a major source of virus variability, and the question of whether novel recombinant viruses would emerge in transgenic plants expressing viral sequences has been a biosafety issue. We describe the results of pyrosequencing the recombinant viral RNAs appearing in transgenic plants expressing the coat protein (CP) gene and 3′ noncoding region of Cucumber mosaic virus RNA3, as well as in nontransgenic controls. The populations of recombinants in both transgenic and nontransgenic plants were similar to those previously described from Sanger sequencing but many more recombinant types were observed, including a novel class of large deletions removing all or nearly the entire CP gene. These results show that populations of recombinant viral genomes arising de novo can be characterized in detail by pyrosequencing, and confirm that the transgenic plants did not harbor novel recombinants of biosafety concern.

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

2020 ◽  
Author(s):  
Piotr Tomasz Bednarek ◽  
Renata Orłowska
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
De Novo ◽  
Culture Conditions ◽  
Ion Concentration ◽  
Tissue Cultures ◽  
Regeneration Medium ◽  
Green Plants ◽  
Anther Cultures

Abstract BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu2+ and Ag+ ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu2+ and Ag+ ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu2+ and Ag+ ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu2+ and Ag+ play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.

scholarly journals ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro

2006 ◽  
Vol 80 (21) ◽  
pp. 10457-10464 ◽  
Author(s):  
Suzanne U. Emerson ◽  
Hanh Nguyen ◽  
Udana Torian ◽  
Robert H. Purcell
Keyword(s):  
Hepatitis E Virus ◽  
Infectious Virus ◽  
Hepatitis E ◽  
Clinical Samples ◽  
Virion Assembly ◽  
Orf3 Protein ◽  
Viral Genomes ◽  
Intracellular Virus

ABSTRACT A subclone of Huh-7 cells that could be relatively efficiently transfected and infected with hepatitis E virus was identified. Following transfection, infectious virus was produced but remained predominantly cell associated. Intracellular virus, recovered by lysis of transfected cells, infected naïve cells. This in vitro-produced virus appeared to be antigenically identical to virus isolated from clinical samples. Lysates from cells transfected with mutant viral genomes unable to synthesize ORF3 protein contained infectious virions that were similar in number, thermostability, and sedimentation characteristics to those in lysates transfected with wild-type viral genomes. Therefore, in contrast to its requirement in vivo, ORF3 protein is not required for infection of Huh-7 cells or production of infectious virus in vitro.

scholarly journals Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

2021 ◽  
Vol 118 (13) ◽  
pp. e2020969118
Author(s):  
Cyril Le Nouën ◽  
Thomas McCarty ◽  
Lijuan Yang ◽  
Michael Brown ◽  
Eckard Wimmer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
De Novo ◽  
Point Mutations ◽  
Surface Glycoprotein ◽  
Temperature Sensitive ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Codon Pair ◽  
Syncytial Virus

Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs—called codon-pair deoptimization (CPD)—provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B–infected cells were encapsidated, expressed high levels of F, and complemented Min B replicationin trans. This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common “defective interfering particles” that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

scholarly journals RNA structure through multidimensional chemical mapping

2016 ◽  
Author(s):  
Siqi Tian ◽  
Rhiju Das
Keyword(s):  
Structure Determination ◽  
Rna Structure ◽  
De Novo ◽  
Compensatory Mutation ◽  
Chemical Mapping ◽  
Rna Molecules ◽  
Viral Genomes ◽  
Rna Interaction

The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed .OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to two hundred nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches, that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-crosslink-map expansion to overcome this and other current limitations to resolving the in vivo "RNA structurome".

scholarly journals Optimization of protoplast regeneration in the model plant Arabidopsis thaliana

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yeong Yeop Jeong ◽  
Hun-Young Lee ◽  
Suk Weon Kim ◽  
Yoo-Sun Noh ◽  
Pil Joon Seo
Keyword(s):  
Tissue Culture ◽  
Arabidopsis Thaliana ◽  
Plant Regeneration ◽  
Tissue Regeneration ◽  
De Novo ◽  
Callus Formation ◽  
Protoplast Regeneration ◽  
Culture Methods ◽  
Cell And Tissue Culture

Abstract Background Plants have a remarkable reprogramming potential, which facilitates plant regeneration, especially from a single cell. Protoplasts have the ability to form a cell wall and undergo cell division, allowing whole plant regeneration. With the growing need for protoplast regeneration in genetic engineering and genome editing, fundamental studies that enhance our understanding of cell cycle re-entry, pluripotency acquisition, and de novo tissue regeneration are essential. To conduct these studies, a reproducible and efficient protoplast regeneration method using model plants is necessary. Results Here, we optimized cell and tissue culture methods for improving protoplast regeneration efficiency in Arabidopsis thaliana. Protoplasts were isolated from whole seedlings of four different Arabidopsis ecotypes including Columbia (Col-0), Wassilewskija (Ws-2), Nossen (No-0), and HR (HR-10). Among these ecotypes, Ws-2 showed the highest potential for protoplast regeneration. A modified thin alginate layer was applied to the protoplast culture at an optimal density of 1 × 106 protoplasts/mL. Following callus formation and de novo shoot regeneration, the regenerated inflorescence stems were used for de novo root organogenesis. The entire protoplast regeneration process was completed within 15 weeks. The in vitro regenerated plants were fertile and produced morphologically normal progenies. Conclusion The cell and tissue culture system optimized in this study for protoplast regeneration is efficient and reproducible. This method of Arabidopsis protoplast regeneration can be used for fundamental studies on pluripotency establishment and de novo tissue regeneration.

scholarly journals Mass Production of Bananas and Plantains (Musa spp.) Plantlets through in vitro Tissue Culture Partway: A Review

2021 ◽  
Vol 2 (4) ◽  
pp. 1-8
Author(s):  
Eustache T. A. E. Agbadje ◽  
Arnaud Agbidinoukoun ◽  
Martine Zandjanakou-Tachin ◽  
Gilles T. H. Cacaï ◽  
Corneille Ahanhanzo
Keyword(s):  
Tissue Culture ◽  
De Novo ◽  
Control Measure ◽  
Shoot Proliferation ◽  
Control Measures ◽  
Direct Organogenesis ◽  
Musa Spp ◽  
In Vitro Tissue Culture ◽  
Planting Material

Bananas and plantains are among the most important food crops in Central and West Africa. Their plantation is lead to many problems. In the recent decades, biotechnology tools using in vitro culture technics are used for the mass and free disease plantlets production in order to increase the bananas production and the yield. The main way of in vitro tissue culture at this end is the direct organogenesis i.e., the ability of plant tissues to form various organs de novo by shoots or roots induction to differentiate from a cell or cell clusters. This review aims to summarize the main results obtained in the organogenesis of bananas and plantains (Musa spp.) under in vitro conditions and to identify the challenges during the process. The research articles used in this review show that micropropagation is a reliable alternative to conventional production system of bananas and plantains planting material. However, the use of the in vitro micropropagation for bananas and plantains entails choosing the optimal explant type and size according to objectives. Benzylaminopurine remains the preferred cytokinin for in vitro banana and plantain shoot proliferation, while the use of thidiazuron appears to be more and more common. Whichever cytokinin used, the optimal cytokinin concentration for shoot proliferation is genotype dependent. This review also focuses on the causes and control measures of the two major banana and plantain micropropagation constraints: lethal tissues browning/darkening and microbial contaminations. It showed that applying the suitable and available control measure, according to the evolution of culture, is necessary. All this available information on the in vitro conditions makes banana and plantain cultivars in vitro organogenesis possible.

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