Limits...
Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

Holmblat B, Jégouic S, Muslin C, Blondel B, Joffret ML, Delpeyroux F - MBio (2014)

Bottom Line: We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A.For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA.Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Biologie des Virus Entériques, Paris, France INSERM U994, Institut National de La Santé et de La Recherche Médicale, Paris, France.

Show MeSH

Related in: MedlinePlus

Evolution of the S2CA17 cl 4.4 viral genome following passaging in cells. (A) Schematic diagram of the genomic structure at passage P3 of the NH-2 recombinant S2CA17 cl 4.4 with a recombination site (solid triangle) in the 3A coding region and an insertion of 70 codons. Dark and light gray shadings indicate sequences from the Sabin 2 and CA17 genomes, respectively. The recombination site (according to Sabin 2 and CA17 numbering, respectively) and the homologous duplicated sequences (indicated by horizontal double-headed arrows) are enlarged, and vertical arrows indicate the 2C/3A cleavage sites. (B and C) At passages P18 and P35, the genomic region harboring the recombination site was amplified by RT-PCR. The amplification product, which was heterogeneous in size (two major bands), was inserted into a plasmid vector, which was then introduced into Escherichia coli. Inserts present in several bacterial plasmids were sequenced. The structures of the recombined regions at passages 18(B) and 35(C) are shown. The 2C and 3A coding regions and recombination site numbering are represented as in panel A. The original structure of the NH-2 parental recombinant S2CA17 cl 4.4 is framed. The lengths of deletions and nucleotides deleted in NH-Δ recombinants are shown. The fortuitous insertion of a VP2 nucleotide fragment into the recombination site of an NH-1 recombinant (2C S2-4980/VP2 S2-1125-1195/2C CA17-5054) is shown. The number of products cloned for each type of recombinant is indicated in brackets. Asterisks indicate genomic rearrangements disrupting the original open reading frame, suggesting the presence of noninfectious viral genomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4128350&req=5

fig4: Evolution of the S2CA17 cl 4.4 viral genome following passaging in cells. (A) Schematic diagram of the genomic structure at passage P3 of the NH-2 recombinant S2CA17 cl 4.4 with a recombination site (solid triangle) in the 3A coding region and an insertion of 70 codons. Dark and light gray shadings indicate sequences from the Sabin 2 and CA17 genomes, respectively. The recombination site (according to Sabin 2 and CA17 numbering, respectively) and the homologous duplicated sequences (indicated by horizontal double-headed arrows) are enlarged, and vertical arrows indicate the 2C/3A cleavage sites. (B and C) At passages P18 and P35, the genomic region harboring the recombination site was amplified by RT-PCR. The amplification product, which was heterogeneous in size (two major bands), was inserted into a plasmid vector, which was then introduced into Escherichia coli. Inserts present in several bacterial plasmids were sequenced. The structures of the recombined regions at passages 18(B) and 35(C) are shown. The 2C and 3A coding regions and recombination site numbering are represented as in panel A. The original structure of the NH-2 parental recombinant S2CA17 cl 4.4 is framed. The lengths of deletions and nucleotides deleted in NH-Δ recombinants are shown. The fortuitous insertion of a VP2 nucleotide fragment into the recombination site of an NH-1 recombinant (2C S2-4980/VP2 S2-1125-1195/2C CA17-5054) is shown. The number of products cloned for each type of recombinant is indicated in brackets. Asterisks indicate genomic rearrangements disrupting the original open reading frame, suggesting the presence of noninfectious viral genomes.

Mentions: The passaging of the NH-2 recombinant S2CA17 cl 4.4 with an insertion of intermediate size (70 codons) led to a mixture of H and NH recombinants at passage 18 in both passage series (Table 1). The genomes of the viruses in the mixture were analyzed in both series. RT-PCR products overlapping recombination sites were inserted into bacterial plasmids for sequencing (190 bacterial clones analyzed). The original 70-codon insert (Fig. 4A) was found in 166 clones, whereas diverse H recombinants with different recombination sites were identified in 20 clones (Fig. 4B). A few clones displayed short insertions and/or deletions and frameshifts, suggesting the presence of nonfunctional genomes. We followed the evolution of the viral population further, by continuing the passaging process until passage 35 (Fig. 4C). We analyzed 43 bacterial clones containing RT-PCR products. The initial duplication (70-codon insert) was retained in 11 clones but lost in the other 32, leading to the generation of homologous recombination sites. Most of the homologous recombination sites were located at sites similar to those previously identified at passage 18, but several new sites were also identified.


Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

Holmblat B, Jégouic S, Muslin C, Blondel B, Joffret ML, Delpeyroux F - MBio (2014)

Evolution of the S2CA17 cl 4.4 viral genome following passaging in cells. (A) Schematic diagram of the genomic structure at passage P3 of the NH-2 recombinant S2CA17 cl 4.4 with a recombination site (solid triangle) in the 3A coding region and an insertion of 70 codons. Dark and light gray shadings indicate sequences from the Sabin 2 and CA17 genomes, respectively. The recombination site (according to Sabin 2 and CA17 numbering, respectively) and the homologous duplicated sequences (indicated by horizontal double-headed arrows) are enlarged, and vertical arrows indicate the 2C/3A cleavage sites. (B and C) At passages P18 and P35, the genomic region harboring the recombination site was amplified by RT-PCR. The amplification product, which was heterogeneous in size (two major bands), was inserted into a plasmid vector, which was then introduced into Escherichia coli. Inserts present in several bacterial plasmids were sequenced. The structures of the recombined regions at passages 18(B) and 35(C) are shown. The 2C and 3A coding regions and recombination site numbering are represented as in panel A. The original structure of the NH-2 parental recombinant S2CA17 cl 4.4 is framed. The lengths of deletions and nucleotides deleted in NH-Δ recombinants are shown. The fortuitous insertion of a VP2 nucleotide fragment into the recombination site of an NH-1 recombinant (2C S2-4980/VP2 S2-1125-1195/2C CA17-5054) is shown. The number of products cloned for each type of recombinant is indicated in brackets. Asterisks indicate genomic rearrangements disrupting the original open reading frame, suggesting the presence of noninfectious viral genomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128350&req=5

fig4: Evolution of the S2CA17 cl 4.4 viral genome following passaging in cells. (A) Schematic diagram of the genomic structure at passage P3 of the NH-2 recombinant S2CA17 cl 4.4 with a recombination site (solid triangle) in the 3A coding region and an insertion of 70 codons. Dark and light gray shadings indicate sequences from the Sabin 2 and CA17 genomes, respectively. The recombination site (according to Sabin 2 and CA17 numbering, respectively) and the homologous duplicated sequences (indicated by horizontal double-headed arrows) are enlarged, and vertical arrows indicate the 2C/3A cleavage sites. (B and C) At passages P18 and P35, the genomic region harboring the recombination site was amplified by RT-PCR. The amplification product, which was heterogeneous in size (two major bands), was inserted into a plasmid vector, which was then introduced into Escherichia coli. Inserts present in several bacterial plasmids were sequenced. The structures of the recombined regions at passages 18(B) and 35(C) are shown. The 2C and 3A coding regions and recombination site numbering are represented as in panel A. The original structure of the NH-2 parental recombinant S2CA17 cl 4.4 is framed. The lengths of deletions and nucleotides deleted in NH-Δ recombinants are shown. The fortuitous insertion of a VP2 nucleotide fragment into the recombination site of an NH-1 recombinant (2C S2-4980/VP2 S2-1125-1195/2C CA17-5054) is shown. The number of products cloned for each type of recombinant is indicated in brackets. Asterisks indicate genomic rearrangements disrupting the original open reading frame, suggesting the presence of noninfectious viral genomes.
Mentions: The passaging of the NH-2 recombinant S2CA17 cl 4.4 with an insertion of intermediate size (70 codons) led to a mixture of H and NH recombinants at passage 18 in both passage series (Table 1). The genomes of the viruses in the mixture were analyzed in both series. RT-PCR products overlapping recombination sites were inserted into bacterial plasmids for sequencing (190 bacterial clones analyzed). The original 70-codon insert (Fig. 4A) was found in 166 clones, whereas diverse H recombinants with different recombination sites were identified in 20 clones (Fig. 4B). A few clones displayed short insertions and/or deletions and frameshifts, suggesting the presence of nonfunctional genomes. We followed the evolution of the viral population further, by continuing the passaging process until passage 35 (Fig. 4C). We analyzed 43 bacterial clones containing RT-PCR products. The initial duplication (70-codon insert) was retained in 11 clones but lost in the other 32, leading to the generation of homologous recombination sites. Most of the homologous recombination sites were located at sites similar to those previously identified at passage 18, but several new sites were also identified.

Bottom Line: We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A.For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA.Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Biologie des Virus Entériques, Paris, France INSERM U994, Institut National de La Santé et de La Recherche Médicale, Paris, France.

Show MeSH
Related in: MedlinePlus