An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA

Deuring, Renate; Klotz, Günther; Doerfler, Walter

doi:10.1073/pnas.78.5.3142

Cited by 69 publications

(38 citation statements)

References 59 publications

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“…First, it was documented that after repeated passaging at high moi on permissive cells with different adenovirus serotypes, subgenomic DNA preferentially containing the left end of the virus genome could be packaged into virions and separated from wild-type virions by CsCl density-gradient centrifugation (17). Second, after repeated high moi passage of Adl2 on human KB cells, hybrid viruses containing symmetrically duplicated chromosomal DNA of KB cell line flanked by 700-1150 bp of DNA fragment from the left terminus of Adl2 (SYREC) were produced (18)(19)(20). These SYREC could be separated from Adl2 by a CsCl equilibrium density gradient and also could be propagated over years together with Adl2.…”

Section: Resultsmentioning

confidence: 99%

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Mitani

Graham

Caskey

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

219

140

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Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression ofviral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (Li, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 106 transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the El region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helperdependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.For several reasons, adenoviruses are attracting increasing attention as expression vectors, especially for human gene therapy (1). First, the virion is relatively stable and can be prepared as a high titer stock (2109 plaque-forming units/ml without purification). Second, adenoviruses can infect nonreplicating cells. Finally, adenovirus vectors have been extensively exploited in vaccine development and proven safe in humans. The current adenovirus vectors have deletions in the El and/or E3 regions, because El proteins can be complemented in 293 cells, the E3 region is dispensable for growth of the virus in cultured cells, and all the other essential viral proteins are encoded in the vector itself. While the lower packaging limit of adenovirus is unknown, the upper packaging limit of adenovirus serotype 5 (Ad5) is -38 kb (2). As a result, vectors with deletions of El and E3 sequences (6 kb deleted in total) have a capacity for inserts of up to -8 kb (25

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Section: Resultsmentioning

confidence: 99%

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Mitani

Graham

Caskey

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

219

140

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“…4,1984 on May 11, 2018 by guest http://mcb.asm.org/ 308 HANAHAN AND GLUZMAN normal right end. Defective adenoviruses have been isolated and shown to stably persist as large palindromes which contain adenoviral information originating only from the left end (6,7). This indicates that the configuration of left-end sequences on both the left and right ends is favorable under certain conditions.…”

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confidence: 99%

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

Hanahan¹,

Gluzman²

1984

Mol. Cell. Biol.

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A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to Clal-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.Human adenovirus type 5 (AdS) has a double-stranded DNA genome of 36 kilobase pairs (kbp) (33). The 5' ends of the viral DNA are covalently linked to a terminal protein through a deoxycytosine-to-serine phosphoryl bond (5, 23). The 55-kilodalton (kDa) terminal protein is a proteolytic cleavage product of an 80-kDa protein which participates in the initiation of DNA replication (2,4,16,26). The terminal protein precursor forms a complex with a 140-kDa adenovirus-encoded polymerase (15, 27), after which the terminal protein precursor becomes modified through its covalent linkage to a deoxycytosine, which will subsequently become the first nucleotide of the daughter strand. During morphogenesis of the virion, the 80-kDa terminal protein precursor is specifically cleaved by an adenovirus-encoded protease to produce the 55-kDa terminal protein (3,26).Isolation of viral DNA commonly includes a nonspecific proteolytic digestion step (with pronase), which removes all but a few amino acids of the terminal protein from the DNA.

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“…Deuring et al describe an Ad12/cellular DNA chimera (symmetric recombinant 1) that consists almost entirely of cellular DNA flanked by 700-1150 bp of the 5Ј end of the Ad12 genome in a large inverted repeat of the type ABCDDЈCЈBЈAЈ. 19 This construct was stable through serial passage for years. Further analysis of a second construct (symmetric recombinant 2) revealed that 2081 bp of the left terminus of Ad12 were linked directly to cellular DNA.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of an adenoviral vector resulting from both homologous and nonhomologous recombination

Smith

Eck

1999

Cancer Gene Ther

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Recombinant adenoviral vectors are being used extensively for gene transfer. During the construction of an E1-deleted virus expressing the human B7-1 gene, an aberrant recombinant (Ad.ihB7-1) arose with an unusual 5Ј sequence. Characterization and sequencing of Ad.ihB7-1 showed that its structure was the result of both homologous and nonhomologous events. The most striking features of the construct were the incorporation of bacterial genomic DNA, an additional inverted terminal repeat, and portions of E1a. The appearance of this construct has implications for vector design and indicates the need for careful analysis and characterization of recombinant adenoviral vectors for clinical use.

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An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA

Abstract: On purification of human adenovirus type 12 (Adl2) by equilibrium sedimentation in CsCl density gradients, two bands of particles, are

Cited by 69 publications

References 59 publications

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

Molecular characterization of an adenoviral vector resulting from both homologous and nonhomologous recombination

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