scholarly journals Two types of non-homologous RNA recombination in brome mosaic virus.

2005 ◽  
Vol 52 (4) ◽  
pp. 833-844 ◽  
Author(s):  
Magdalena Alejska ◽  
Nelli Malinowska ◽  
Anna Urbanowicz ◽  
Marek Figlerowicz
Keyword(s):  
Mosaic Virus ◽  
Genetic Recombination ◽  
Genetic Material ◽  
Brome Mosaic Virus ◽  
Homologous Sequence ◽  
Template Switching ◽  
Rna Recombination ◽  
Genomic Rnas ◽  
Site Specific ◽  
Rna Interaction

Non-homologous RNA recombination is a process enabling the exchange of genetic material between various (related or unrelated) RNA-based viruses. Despite extensive investigations its molecular mechanism remains unclear. Studies on genetic recombination in brome mosaic virus (BMV) have shown that local hybridization between genomic RNAs induces frequent non-homologous crossovers. A detailed analysis of recombinant structures suggested that local complementary regions might be involved in two types of non-homologous recombination in BMV: site-specific and heteroduplex-mediated. To verify the above hypothesis and better recognize the mechanism of the phenomenon studied we have tested how the putative types of recombination are affected by a specific mutation in the BMV polymerase gene or by changes in RNA structure. The experiments undertaken revealed substantial differences between site-specific and heteroduplex-mediated recombination, indicating that they occur according to different mechanisms. The former can be classified as homology-assisted, and the latter as homology-independent. In addition to local RNA/RNA hybridization, short regions of homology are required for site-specific crossovers to occur. They are most efficiently mediated if one homologous sequence is located at the beginning of and the second just before a double-stranded region. At present it is difficult to state what is the mechanism of heteroduplex-mediated recombination. Earlier it was postulated that strong RNA/RNA interaction enforces template switching by the viral replicase. There are, however, several observations questioning this model and indicating that some other factors, which are still unknown, may influence heteroduplex-mediated crossovers.

scholarly journals RNA recombination in brome mosaic virus, a model plus strand RNA virus.

1998 ◽  
Vol 45 (4) ◽  
pp. 847-868 ◽  
Author(s):  
M Figlerowicz ◽  
J J Bujarski
Keyword(s):  
Mosaic Virus ◽  
Genetic Recombination ◽  
Rna Virus ◽  
Brome Mosaic Virus ◽  
Template Switching ◽  
Rna Recombination ◽  
Signal Sequences ◽  
Experimental Systems ◽  
Replicase Complex ◽  
Molecular Aspects

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.

scholarly journals Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

2016 ◽  
Vol 90 (17) ◽  
pp. 7748-7760 ◽  
Author(s):  
Haley S. Hoover ◽  
Joseph Che-Yen Wang ◽  
Stefani Middleton ◽  
Peng Ni ◽  
Adam Zlotnick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Mosaic Virus ◽  
Posttranslational Modifications ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Genomic Rnas ◽  
The Stability ◽  
Rna Interaction

ABSTRACTThe four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.IMPORTANCEHow icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

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 How RNA viruses exchange their genetic material.

2001 ◽  
Vol 48 (2) ◽  
pp. 391-407 ◽  
Author(s):  
M Alejska ◽  
A Kurzyńska-Kokorniak ◽  
M Broda ◽  
R Kierzek ◽  
M Figlerowicz
Keyword(s):  
Rna Structure ◽  
Rna Viruses ◽  
Genetic Recombination ◽  
Rna Virus ◽  
Genetic Material ◽  
Brome Mosaic Virus ◽  
Special Importance ◽  
Rna Recombination ◽  
Nonhomologous Recombination

One of the most unusual features of RNA viruses is their enormous genetic variability. Among the different processes contributing to the continuous generation of new viral variants RNA recombination is of special importance. This process has been observed for human, animal, plant and bacterial viruses. The collected data reveal a great susceptibility of RNA viruses to recombination. They also indicate that genetic RNA recombination (especially the nonhomologous one) is a major factor responsible for the emergence of new viral strains or species. Although the formation and accumulation of viral recombinants was observed in numerous RNA viruses, the molecular basis of this phenomenon was studied in only a few viral species. Among them, brome mosaic virus (BMV), a model (+)RNA virus offers the best opportunities to investigate various aspects of genetic RNA recombination in vivo. Unlike any other, the BMV-based system enables homologous and nonhomologous recombination studies at both the protein and RNA levels. As a consequence, BMV is the virus for which the structural requirements for genetic RNA recombination have been most precisely established. Nevertheless, the previously proposed model of genetic recombination in BMV still had one weakness: it could not really explain the role of RNA structure in nonhomologous recombination. Recent discoveries concerning the latter problem give us a chance to fill this gap. That is why in this review we present and thoroughly discuss all results concerning nonhomologous recombination in BMV that have been obtained until now.

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.

cDNA cloning and in vitro transcription of the complete brome mosaic virus genome

1984 ◽  
Vol 4 (12) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Ahlquist ◽  
M Janda
Keyword(s):  
Mosaic Virus ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
In Vitro Transcription ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
In Vitro Translation ◽  
In Vitro Production ◽  
Genomic Rnas

Complete cDNA copies of each of the brome mosaic virus genomic RNAs (3.2, 2.8, and 2.1 kilobases in length) were cloned in a novel transcription vector, pPM1, designed to provide exact control of the transcription initiation site. After cleavage at a unique EcoRI site immediately downstream of the inserted cDNA, these clones can be transcribed in vitro by Escherichia coli RNA polymerase to yield complete copies of the brome mosaic virus RNAs. Dideoxy sequencing of 5' transcript cDNA runoff products and direct sequencing of 32P-3'-end-labeled transcripts show that such transcripts initiate at the same 5' position as natural viral RNA and terminate within the EcoRI runoff site after copying the entire viral RNA sequence. When synthesized in the presence of m7GpppG, the transcripts bear the natural capped 5' terminus of brome mosaic virus RNAs. Such transcripts direct the in vitro translation of proteins which coelectrophorese with the translation products of natural brome mosaic virus RNAs. pPM1 should facilitate in vitro production of other viral and nonviral RNAs.

scholarly journals Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

2019 ◽  
Author(s):  
Antara Chakravarty ◽  
Christian Beren ◽  
Rees Garmann ◽  
A.L.N. Rao
Keyword(s):  
Mosaic Virus ◽  
Transient Expression ◽  
Rna Binding ◽  
Conformational Dynamics ◽  
Brome Mosaic Virus ◽  
Crystallographic Structure ◽  
Binding Motif ◽  
Genomic Rnas ◽  
Viral Capsids

ABSTRACTViral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replicated three-molecule RNA progeny of Brome mosaic virus (BMV) are packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Type 1 (B1v) and type 2 (B2v) virions respectively package genomic RNA1 or RNA2, while type 3 (B3+4v) co-packages genomic RNA3 (B3) and its sub-genomic RNA4 (B4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Physical and biochemical approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types, so protease-based mapping experiments were used to analyze the conformational dynamics of the individual virions. The crystallographic structure of the BMV capsid shows four trypsin-cleavage sites (K65, R103, K111 and K165 on the A, B and C subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1v and B2v released only two peptides involving amino acids 2-8 and 16-22 from the N-proximal arginine-rich RNA binding motif. In contrast, B3+4v capsids are unstable to trypsin, releasing several peptides in addition to the four sites predicted to be exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCEThe majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that in solution virions are highly dynamic assemblies. The three genomic RNAs (RNAs 1, 2 and 3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the capsid dynamics by MALDI-TOF analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNAs1 and 2 are more stable and dynamically distinct from those co-packaging RNA3 and 4, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

scholarly journals Evidence for RNA recombination between distinct isolates of Pepino mosaic virus.

2010 ◽  
Vol 57 (3) ◽  
Author(s):  
Beata Hasiów-Jaroszewska ◽  
Arnold Kuzniar ◽  
Sander A Peters ◽  
Jack A M Leunissen ◽  
Henryk Pospieszny
Keyword(s):  
Mosaic Virus ◽  
Genetic Recombination ◽  
Genomic Sequences ◽  
Rna Recombination ◽  
Pepino Mosaic Virus ◽  
Bioinformatics Tools ◽  
Genome Wide ◽  
Virus Genomes ◽  
Double Recombinant

Genetic recombination plays an important role in the evolution of virus genomes. In this study we analyzed publicly available genomic sequences of Pepino mosaic virus (PepMV) for recombination events using several bioinformatics tools. The genome-wide analyses not only confirm the presence of previously found recombination events in PepMV but also provide the first evidence for double recombinant origin of the US2 isolate.

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