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 Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

1998 ◽  
Vol 72 (2) ◽  
pp. 1122-1130 ◽  
Author(s):  
Peter D. Nagy ◽  
Jozef J. Bujarski
Keyword(s):  
Homologous Recombination ◽  
Mosaic Virus ◽  
Hot Spots ◽  
Rna Virus ◽  
Hot Spot ◽  
Brome Mosaic Virus ◽  
Complex Nature ◽  
Template Switching ◽  
Rna Recombination ◽  
Recombination Hot Spots

ABSTRACT It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.

scholarly journals The AU-Rich RNA Recombination Hot Spot Sequence of Brome Mosaic Virus Is Functional in Tombusviruses: Implications for the Mechanism of RNA Recombination

2004 ◽  
Vol 78 (5) ◽  
pp. 2288-2300 ◽  
Author(s):  
Natalia Shapka ◽  
Peter D. Nagy
Keyword(s):  
Mosaic Virus ◽  
Hot Spots ◽  
Hot Spot ◽  
Brome Mosaic Virus ◽  
Template Switching ◽  
Rna Recombination ◽  
Replicase Proteins ◽  
Recombination Hot Spots ◽  
Recombination Junction ◽  
Selection Of

ABSTRACT RNA recombination can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase “landing” during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.

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 Homologous Crossovers among Molecules of Brome Mosaic Bromovirus RNA1 or RNA2 Segments In Vivo

2005 ◽  
Vol 79 (9) ◽  
pp. 5732-5742 ◽  
Author(s):  
Anna Urbanowicz ◽  
Magdalena Alejska ◽  
Piotr Formanowicz ◽  
Jacek Błażewicz ◽  
Marek Figlerowicz ◽  
...  
Keyword(s):  
Hot Spot ◽  
Rna Replication ◽  
Viral Rna ◽  
Noncoding Regions ◽  
Genome Variability ◽  
Coding Regions ◽  
Virus Life Cycle ◽  
Brome Mosaic Bromovirus ◽  
Rna Genome

ABSTRACT Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3′ and 5′ noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.

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 Efficient Homologous RNA Recombination and Requirement for an Open Reading Frame during Replication of Equine Arteritis Virus Defective Interfering RNAs

2000 ◽  
Vol 74 (19) ◽  
pp. 9062-9070 ◽  
Author(s):  
Richard Molenkamp ◽  
Sophie Greve ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Virus ◽  
Rna Replication ◽  
Proximal Region ◽  
Full Length ◽  
Equine Arteritis Virus ◽  
Open Reading Frame ◽  
Replicase Gene ◽  
Rna Recombination ◽  
Reading Frame ◽  
A Genome

ABSTRACT Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156–3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3′- and 5′-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5′-proximal region was found to be approximately 100-fold lower than that in the 3′-proximal part of the genome.

scholarly journals Bacterial RecA Protein Promotes Adenoviral Recombination duringIn VitroInfection

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Jeong Yoon Lee ◽  
Ji Sun Lee ◽  
Emma C. Materne ◽  
Rahul Rajala ◽  
Ashrafali M. Ismail ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Hot Spots ◽  
Genetic Recombination ◽  
Reca Protein ◽  
Human Adenovirus ◽  
Penton Base ◽  
Content Type ◽  
E Coli ◽  
Recombination Hot Spots ◽  
Loop 2

ABSTRACTAdenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of anEscherichia colilysate increased recombination; this was blocked in a RecA mutant strain,E. coliDH5α, or upon RecA depletion. Recombination increased in the presence ofE. colilysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiADsequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCEAdenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

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 Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots

2016 ◽  
Vol 37 (3) ◽  
Author(s):  
Irina V. Getun ◽  
Zhen Wu ◽  
Mohammad Fallahi ◽  
Souad Ouizem ◽  
Qin Liu ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Meiotic Recombination ◽  
Hot Spots ◽  
Specific Binding ◽  
Hot Spot ◽  
Histone H3 ◽  
Open Chromatin ◽  
Histone Marks ◽  
Functional Roles ◽  
Recombination Hot Spots

ABSTRACT Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined “hot spots.” In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots.

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