scholarly journals Co-infection with two strains of Brome mosaic bromovirus reveals common RNA recombination sites in different hosts

2015 ◽  
Vol 1 (1) ◽  
pp. vev021 ◽  
Author(s):  
Beivy Kolondam ◽  
Parth Rao ◽  
Joanna Sztuba-Solinska ◽  
Philipp H. Weber ◽  
Aleksandra Dzianott ◽  
...  
Keyword(s):  
Rna Recombination ◽  
Brome Mosaic Bromovirus

scholarly journals Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus

2006 ◽  
Vol 80 (12) ◽  
pp. 6182-6187 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Recombination Frequency ◽  
In Vivo Studies ◽  
Rna Recombination ◽  
In Vitro System ◽  
Brome Mosaic Bromovirus ◽  
Subgenomic Promoter

ABSTRACT Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78:8552-8564, 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (−)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.

scholarly journals RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts

2002 ◽  
Vol 76 (24) ◽  
pp. 12654-12662 ◽  
Author(s):  
R. C. L. Olsthoorn ◽  
A. Bruyere ◽  
A. Dzianott ◽  
J. J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Experimental Evidence ◽  
Landing Site ◽  
Brome Mosaic Virus ◽  
Model System ◽  
Rna Recombination ◽  
Stem Loop ◽  
Noncoding Regions ◽  
Negative Strand ◽  
Brome Mosaic Bromovirus

ABSTRACT A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3′ noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.

scholarly journals Frequent Homologous Recombination Events between Molecules of One RNA Component in a Multipartite RNA Virus

2000 ◽  
Vol 74 (9) ◽  
pp. 4214-4219 ◽  
Author(s):  
A. Bruyere ◽  
M. Wantroba ◽  
S. Flasinski ◽  
A. Dzianott ◽  
J. J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Rna Virus ◽  
Hot Spot ◽  
Rna Replication ◽  
Rna Recombination ◽  
Brome Mosaic Bromovirus ◽  
Local Lesion Host ◽  
Minor Sequence ◽  
Recombination Hot Spots

ABSTRACT Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

scholarly journals A Transcriptionally Active Subgenomic Promoter Supports Homologous Crossovers in a Plus-Strand RNA Virus

2003 ◽  
Vol 77 (12) ◽  
pp. 6769-6776 ◽  
Author(s):  
Rafal Wierzchoslawski ◽  
Aleksandra Dzianott ◽  
Selvi Kunimalayan ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Molecular Mechanism ◽  
Rna Virus ◽  
Viral Evolution ◽  
Rna Recombination ◽  
Recombination Activity ◽  
Brome Mosaic Bromovirus ◽  
Restriction Sites ◽  
Subgenomic Promoter

ABSTRACT Genetic RNA recombination plays an important role in viral evolution, but its molecular mechanism is not well understood. In this work we describe homologous RNA recombination activity that is supported by a subgenomic promoter (sgp) region in the RNA3 segment of brome mosaic bromovirus (BMV), a tripartite plus-strand RNA virus. The crossover frequencies were determined by coinoculations with pairs of BMV RNA3 variants that carried a duplicated sgp region flanked by marker restriction sites. A region composed of the sgp core, a poly(A) tract, and an upstream enhancer supported homologous exchanges in 25% of the analyzed RNA3 progeny. However, mutations in the sgp core stopped both the transcription of the sgp RNA and homologous recombination. These data provide evidence for an association of RNA recombination with transcription.

scholarly journals Norovirus recombination

2007 ◽  
Vol 88 (12) ◽  
pp. 3347-3359 ◽  
Author(s):  
Rowena A. Bull ◽  
Mark M. Tanaka ◽  
Peter A. White
Keyword(s):  
High Frequency ◽  
Driving Force ◽  
Recombination Event ◽  
Viral Evolution ◽  
Rna Recombination ◽  
Recombination Point ◽  
The Family ◽  
Double Recombination ◽  
Double Recombination Event ◽  
Recombination Breakpoints

RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV) recombinants and they are now reported at high frequency. Currently, there is no classification system for recombinant NoVs and a widely accepted recombinant genotyping system is still needed. Consequently, there is duplication in reporting of novel recombinants. This has led to difficulties in defining the number and types of recombinants in circulation. In this study, 120 NoV nucleotide sequences were compiled from the current GenBank database and published literature. NoV recombinants and their recombination breakpoints were identified using three methods: phylogenetic analysis, SimPlot analysis and the maximum χ 2 method. A total of 20 NoV recombinant types were identified in circulation worldwide. The recombination point is the ORF1/2 overlap in all isolates except one, which demonstrated a double recombination event within the polymerase region.

scholarly journals Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs

1992 ◽  
Vol 20 (13) ◽  
pp. 3375-3381 ◽  
Author(s):  
Robbert G. van der Most ◽  
Leo Heijnen ◽  
Willy J.M. Spaan ◽  
Raoul J. de Groot
Keyword(s):  
Rna Recombination ◽  
Site Specific

scholarly journals Coronaviruses as Vectors: Stability of Foreign Gene Expression

2005 ◽  
Vol 79 (20) ◽  
pp. 12742-12751 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Bert Jan Haijema ◽  
David Boss ◽  
Frank W. H. Heuts ◽  
Peter J. M. Rottier
Keyword(s):  
Genetic Stability ◽  
Vaccine Development ◽  
Hepatitis Virus ◽  
Firefly Luciferase ◽  
Foreign Gene ◽  
Rna Recombination ◽  
Large Deletions ◽  
Group 2 ◽  
Attenuated Viruses ◽  
Foreign Genes

ABSTRACT Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.

scholarly journals RNA Recombination Plays a Major Role in Genomic Change during Circulation of Coxsackie B Viruses

2004 ◽  
Vol 78 (6) ◽  
pp. 2948-2955 ◽  
Author(s):  
M. Steven Oberste ◽  
Silvia Peñaranda ◽  
Mark A. Pallansch
Keyword(s):  
Phylogenetic Trees ◽  
Clinical Isolates ◽  
Human Enterovirus ◽  
Rna Recombination ◽  
Genomic Change ◽  
Genome Region ◽  
Nontranslated Region ◽  
Frequent Event ◽  
Coxsackie B Viruses ◽  
Coxsackie B

ABSTRACT RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5′-nontranslated region (5′-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, ∼320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5′-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.

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