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Mutational analysis of the avian adenovirus CELO, which provides a basis for gene delivery vectors.

ABSTRACT: The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.

SUBMITTER: Michou AI

PROVIDER: S-EPMC103964 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Mutational analysis of the avian adenovirus CELO, which provides a basis for gene delivery vectors.

Michou A I AI Lehrmann H H Saltik M M Cotten M M

Journal of virology 19990201 2

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted b ...[more]

PMID: 9882345

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Project description:BACKGROUND:Retrotransposons are abundant components of plant genomes, and although some plant retrotransposons have been used as insertional mutagens, these mobile genetic elements have not been widely exploited for plant genome manipulation. In vertebrates and yeast, retrotransposons and retroviruses are routinely altered to carry additional genes that are copied into complementary (c)DNA through reverse transcription. Integration of cDNA results in gene delivery; recombination of cDNA with homologous chromosomal sequences can create targeted gene modifications. Plant retrotransposon-based vectors, therefore, may provide new opportunities for plant genome engineering. RESULTS:A retrotransposon vector system was developed for gene delivery in plants based on the Tnt1 element from Nicotiana tabacum. Mini-Tnt1 transfer vectors were constructed that lack coding sequences yet retain the 5' and 3' long terminal repeats (LTRs) and adjacent cis sequences required for reverse transcription. The internal coding region of Tnt1 was replaced with a neomycin phosphotransferase gene to monitor replication by reverse transcription. Two different mini-Tnt1 s were developed: one with the native 5' LTR and the other with a chimeric 5' LTR that had the first 233 bp replaced by the CaMV 35 S promoter. After transfer into tobacco protoplasts, both vectors undergo retrotransposition using GAG and POL proteins provided in trans by endogenous Tnt1 elements. The transposition frequencies of mini-Tnt1 vectors are comparable with native Tnt1 elements, and like the native elements, insertion sites are within or near coding sequences. In this paper, we provide evidence that template switching occurs during mini-Tnt1 reverse transcription, indicating that multiple copies of Tnt1 mRNA are packaged into virus-like particles. CONCLUSIONS:Our data demonstrate that mini-Tnt1 vectors can replicate efficiently in tobacco cells using GAG and POL proteins provided in trans by native Tnt1 elements. This suggests that helper Tnt1 constructs can be developed to enable a Tnt1-based two-component vector system that could be used in other plant species. Such a vector system may prove useful for gene delivery or the production of cDNA that can serve as a donor molecule for gene modification through homologous recombination.

Project description:Following receptor binding and internalization, intracellular trafficking of adenovirus (Ad) among subgroups B and C is different, with significant amounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly translocate to the nucleus. To evaluate the role of the fiber in these differences, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimeric vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fiber) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated viruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly translocated to the nucleus (within 1 h of infection), while Ad7 virions were widely distributed in the cytoplasm at the same time point. Interestingly, chimeric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h following infection were 72% +/- 4%, 32% +/- 6%, and 38% +/- 2%, respectively. Consistent with these observations, fluorescence in situ hybridization demonstrated that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucleus and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demonstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 DNA at 1 h postinfection were 80% +/- 13% and 43% +/- 1%, respectively. Although it has been generally accepted that Ad fiber protein mediates attachment of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surface, fiber likely modulates intracellular trafficking as well.

Dataset Information

Mutational analysis of the avian adenovirus CELO, which provides a basis for gene delivery vectors.

Publications

Mutational analysis of the avian adenovirus CELO, which provides a basis for gene delivery vectors.

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