Limits...
The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection.

Shi BJ, Symons RH, Palukaitis P - Nucleic Acids Res. (2007)

Bottom Line: The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs.A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site.The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Plant Functional Genomics, University of Adelaide, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia. bujun.shi@acpfg.com.au

ABSTRACT
RNA-RNA recombination is an important pathway in virus evolution and has been described for many viruses. However, the factors driving recombination or promoting the selection of recombinants are still unclear. Here, we show that the small movement protein (2b) was able to promote selection of RNA 1/2-RNA 3 recombinants within a chimeric virus having RNAs 1 and 2 from cucumber mosaic virus, and RNA 3 from the related tomato aspermy virus, along with heterologous 2b genes. The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs. The nature of the RNA 3 also influenced the selection of the recombinant RNAs. A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site. The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis.

Show MeSH

Related in: MedlinePlus

Genome organization of C1C2T2BT3Δ163(A), C1C2T2BT3Δ163(G), C1C2W2BT3Δ163(A), C1C2W2BT3Δ163(G) and their derived recombinant viruses. The origin of the 3′ NTR of each RNA 3 is differentiated by fill patterns (stippled for RNA 1 and crosshatched for RNA 2). The short black bars on the RNAs represent the 23 nt of sequence identity. The arrows on RNA 3 represent one of the tandem repeats (A or G). The junction site of each RNA 3 recombinant is indicated. The nucleotide sequence in the junction site of the RNA 3 recombinant derived from C1C2W2BT3Δ163(G) is indicated. The sequence in italics is from TAV RNA 3. The sequences underlined represent the imperfect 19-nt repeats. The single nucleotide difference in the repeats is indicated by an asterisk.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275080&req=5

Figure 5: Genome organization of C1C2T2BT3Δ163(A), C1C2T2BT3Δ163(G), C1C2W2BT3Δ163(A), C1C2W2BT3Δ163(G) and their derived recombinant viruses. The origin of the 3′ NTR of each RNA 3 is differentiated by fill patterns (stippled for RNA 1 and crosshatched for RNA 2). The short black bars on the RNAs represent the 23 nt of sequence identity. The arrows on RNA 3 represent one of the tandem repeats (A or G). The junction site of each RNA 3 recombinant is indicated. The nucleotide sequence in the junction site of the RNA 3 recombinant derived from C1C2W2BT3Δ163(G) is indicated. The sequence in italics is from TAV RNA 3. The sequences underlined represent the imperfect 19-nt repeats. The single nucleotide difference in the repeats is indicated by an asterisk.

Mentions: To determine the nature of the recombinant RNA 3s derived from the 163-nt repeat-deletion mutants, we again purified the recombinant RNA 3s of C1C2T2BT3Δ163(A), C1C2W2BT3Δ163(A), C1C2T2BT3Δ163(G) and C1C2W2BT3Δ163(G), used RT-PCR to amplify the recombinant sequences, cloned the RT-PCR products and sequenced them. The sequences of the recombinant RNA 3 derived from C1C2T2BT3Δ163(A) contained the 5′-proximal 2082 nt from TAV RNA 3 and the 3′ proximal 310-nt 3′ NTR of CMV RNA 1 (Figure 5A). The CMV RNA 1 NTR sequence was identical to that present in the recombinant RNA 3 derived from C1C2T2BT3. However, the TAV RNA 3 sequence was 22 nt longer than that of the recombinant RNA 3 derived from C1C2T2BT3 (compare Figures 3A and 5A), suggesting that the first 163-nt repeat might have a role in determining the crossover site on one of the participating RNAs, despite not affecting the crossover site on the other participating RNA. By contrast, the sequence of the recombinant RNA 3 derived from C1C2T2BT3Δ163(G) was exactly the same as that derived from C1C2T2BT3 (compare Figures 3A and 5B), indicating that the second 163-nt repeat had no role in determining the crossover sites on either RNA.Figure 5.


The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection.

Shi BJ, Symons RH, Palukaitis P - Nucleic Acids Res. (2007)

Genome organization of C1C2T2BT3Δ163(A), C1C2T2BT3Δ163(G), C1C2W2BT3Δ163(A), C1C2W2BT3Δ163(G) and their derived recombinant viruses. The origin of the 3′ NTR of each RNA 3 is differentiated by fill patterns (stippled for RNA 1 and crosshatched for RNA 2). The short black bars on the RNAs represent the 23 nt of sequence identity. The arrows on RNA 3 represent one of the tandem repeats (A or G). The junction site of each RNA 3 recombinant is indicated. The nucleotide sequence in the junction site of the RNA 3 recombinant derived from C1C2W2BT3Δ163(G) is indicated. The sequence in italics is from TAV RNA 3. The sequences underlined represent the imperfect 19-nt repeats. The single nucleotide difference in the repeats is indicated by an asterisk.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275080&req=5

Figure 5: Genome organization of C1C2T2BT3Δ163(A), C1C2T2BT3Δ163(G), C1C2W2BT3Δ163(A), C1C2W2BT3Δ163(G) and their derived recombinant viruses. The origin of the 3′ NTR of each RNA 3 is differentiated by fill patterns (stippled for RNA 1 and crosshatched for RNA 2). The short black bars on the RNAs represent the 23 nt of sequence identity. The arrows on RNA 3 represent one of the tandem repeats (A or G). The junction site of each RNA 3 recombinant is indicated. The nucleotide sequence in the junction site of the RNA 3 recombinant derived from C1C2W2BT3Δ163(G) is indicated. The sequence in italics is from TAV RNA 3. The sequences underlined represent the imperfect 19-nt repeats. The single nucleotide difference in the repeats is indicated by an asterisk.
Mentions: To determine the nature of the recombinant RNA 3s derived from the 163-nt repeat-deletion mutants, we again purified the recombinant RNA 3s of C1C2T2BT3Δ163(A), C1C2W2BT3Δ163(A), C1C2T2BT3Δ163(G) and C1C2W2BT3Δ163(G), used RT-PCR to amplify the recombinant sequences, cloned the RT-PCR products and sequenced them. The sequences of the recombinant RNA 3 derived from C1C2T2BT3Δ163(A) contained the 5′-proximal 2082 nt from TAV RNA 3 and the 3′ proximal 310-nt 3′ NTR of CMV RNA 1 (Figure 5A). The CMV RNA 1 NTR sequence was identical to that present in the recombinant RNA 3 derived from C1C2T2BT3. However, the TAV RNA 3 sequence was 22 nt longer than that of the recombinant RNA 3 derived from C1C2T2BT3 (compare Figures 3A and 5A), suggesting that the first 163-nt repeat might have a role in determining the crossover site on one of the participating RNAs, despite not affecting the crossover site on the other participating RNA. By contrast, the sequence of the recombinant RNA 3 derived from C1C2T2BT3Δ163(G) was exactly the same as that derived from C1C2T2BT3 (compare Figures 3A and 5B), indicating that the second 163-nt repeat had no role in determining the crossover sites on either RNA.Figure 5.

Bottom Line: The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs.A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site.The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Plant Functional Genomics, University of Adelaide, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia. bujun.shi@acpfg.com.au

ABSTRACT
RNA-RNA recombination is an important pathway in virus evolution and has been described for many viruses. However, the factors driving recombination or promoting the selection of recombinants are still unclear. Here, we show that the small movement protein (2b) was able to promote selection of RNA 1/2-RNA 3 recombinants within a chimeric virus having RNAs 1 and 2 from cucumber mosaic virus, and RNA 3 from the related tomato aspermy virus, along with heterologous 2b genes. The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs. The nature of the RNA 3 also influenced the selection of the recombinant RNAs. A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site. The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis.

Show MeSH
Related in: MedlinePlus