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A novel method for generating and screening peptides and libraries displayed on adenovirus fiber.

Lupold SE, Kudrolli TA, Chowdhury WH, Wu P, Rodriguez R - Nucleic Acids Res. (2007)

Bottom Line: Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology.The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene.For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

View Article: PubMed Central - PubMed

Affiliation: James Buchanan Brady Urology Institute, Johns Hopkins University School of Medicine, Broadway Research Building 467, 733N Broadway, Baltimore, MD 21205, USA. slupold@jhmi.edu

ABSTRACT
Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre-lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

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Related in: MedlinePlus

Schematic for generating, selecting and rescuing adenoviral peptide libraries. Clockwise from top left: CAR-ablated fiber-peptide library cassettes are unidirectionally shuttled into fiber-less pFex viral genomes in 293cre57 cells. Some of the resulting CAR detargeted and peptide retargeted viruses infect the target cells. Resulting viral plaques can be isolated and amplified. The floxed fiber cassette can be amplified by PCR and recombined with RPuc-Rescue in 294cre E. coli. Growth in appropriate antibiotics and 5% sucrose selects new fiber shuttles that are directly applicable to sequencing, further modification or recombination to generate new virus in plasmid or viral form.
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Figure 5: Schematic for generating, selecting and rescuing adenoviral peptide libraries. Clockwise from top left: CAR-ablated fiber-peptide library cassettes are unidirectionally shuttled into fiber-less pFex viral genomes in 293cre57 cells. Some of the resulting CAR detargeted and peptide retargeted viruses infect the target cells. Resulting viral plaques can be isolated and amplified. The floxed fiber cassette can be amplified by PCR and recombined with RPuc-Rescue in 294cre E. coli. Growth in appropriate antibiotics and 5% sucrose selects new fiber shuttles that are directly applicable to sequencing, further modification or recombination to generate new virus in plasmid or viral form.

Mentions: Viral burst from each sample were quantified 48 h post-infection. In comparison to the wild-type control, the FBR2-6X library produced ∼0.075% of the number of bursts. There were no infected cells bursts in the negative (N541S) control. Individual plaques were randomly isolated and amplified in 24-well plates. Aliquots were taken for PCR amplification of the floxed fiber cassette. The resulting PCR product was then recombined with RPuc-Rescue, an acceptor plasmid that contains half-mutant lox sites flanking a SacB gene for sucrose selection, in 294cre E. coli (Figure 5). Once recombined into RPuc-Rescue, the cloned fiber gene is available for sequencing, further modification, or recombination with pFex viral genomes to create a subsequent virus.Figure 5.


A novel method for generating and screening peptides and libraries displayed on adenovirus fiber.

Lupold SE, Kudrolli TA, Chowdhury WH, Wu P, Rodriguez R - Nucleic Acids Res. (2007)

Schematic for generating, selecting and rescuing adenoviral peptide libraries. Clockwise from top left: CAR-ablated fiber-peptide library cassettes are unidirectionally shuttled into fiber-less pFex viral genomes in 293cre57 cells. Some of the resulting CAR detargeted and peptide retargeted viruses infect the target cells. Resulting viral plaques can be isolated and amplified. The floxed fiber cassette can be amplified by PCR and recombined with RPuc-Rescue in 294cre E. coli. Growth in appropriate antibiotics and 5% sucrose selects new fiber shuttles that are directly applicable to sequencing, further modification or recombination to generate new virus in plasmid or viral form.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175307&req=5

Figure 5: Schematic for generating, selecting and rescuing adenoviral peptide libraries. Clockwise from top left: CAR-ablated fiber-peptide library cassettes are unidirectionally shuttled into fiber-less pFex viral genomes in 293cre57 cells. Some of the resulting CAR detargeted and peptide retargeted viruses infect the target cells. Resulting viral plaques can be isolated and amplified. The floxed fiber cassette can be amplified by PCR and recombined with RPuc-Rescue in 294cre E. coli. Growth in appropriate antibiotics and 5% sucrose selects new fiber shuttles that are directly applicable to sequencing, further modification or recombination to generate new virus in plasmid or viral form.
Mentions: Viral burst from each sample were quantified 48 h post-infection. In comparison to the wild-type control, the FBR2-6X library produced ∼0.075% of the number of bursts. There were no infected cells bursts in the negative (N541S) control. Individual plaques were randomly isolated and amplified in 24-well plates. Aliquots were taken for PCR amplification of the floxed fiber cassette. The resulting PCR product was then recombined with RPuc-Rescue, an acceptor plasmid that contains half-mutant lox sites flanking a SacB gene for sucrose selection, in 294cre E. coli (Figure 5). Once recombined into RPuc-Rescue, the cloned fiber gene is available for sequencing, further modification, or recombination with pFex viral genomes to create a subsequent virus.Figure 5.

Bottom Line: Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology.The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene.For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

View Article: PubMed Central - PubMed

Affiliation: James Buchanan Brady Urology Institute, Johns Hopkins University School of Medicine, Broadway Research Building 467, 733N Broadway, Baltimore, MD 21205, USA. slupold@jhmi.edu

ABSTRACT
Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre-lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

Show MeSH
Related in: MedlinePlus