Genome Structure of Adenovirus 12 Host Range Mutants Adapted to Growth in Simian Vero Cells

Abstract

Mutants of adenovirus type 12 adapted to growth in Vero cells were studied by restriction enzyme and sequence analysis. Plaque-purified mutants were isolated either after long-term passage of the virus in Vero cells (CS-1, CL-1) or from Vero cells transfected with viral DNA (11-g, 12-k). The plaque-forming activity of these mutants was equal or even slightly higher in Vero cells than in HeLa cells, whereas the activity of wild-type virus in HeLa cells exceeded that in Vero cells by about one to two orders of magnitude. All mutants tested had an insertion of variable length consisting of adenoviral sequences at the right end of the viral genome. Sizes of the insertions were determined to be 294 bp (CS-1,12-k), 560 (11-g), or 180 bp (CL-1). Additional insertions of multiple size and less than equimolar amount were detected and seem to exist in some of the viral particles. Furthermore, all four mutants tested showed an identical 69-bp deletion in the first exon of the Ela gene.

The Transformation-Defective Adenovirus 12 Host Range Mutant CS-1 Lacks the Ela-Specific 9.5S mRNA and Contains a Second Deletion in E1b

“…The adenovirus 12 (Adl2) host range mutant Adl2CS-l (CS-1) is adapted to efficient replica tion in simian Vero cells which are only semipermissive for Adl2 [1,2], Moreover, this mutant is not or extremely weakly oncogenic in newborn hamsters [3]. Genome analysis had so far shown two alternations: additional viral sequences at the right terminus and a deletion of 69 bp in the first exon of the Ela gene [2], For further analysis of the deleted Ela gene, we used the cDNA polymerase chain reaction (PCR) technique [4] Fig-1-Ela specific cDNA PCR products from Adl2wt-and CS-l-infected HeLa and Vero cells resolved on a 2% agarose gel.…”

“…Genome analysis had so far shown two alternations: additional viral sequences at the right terminus and a deletion of 69 bp in the first exon of the Ela gene [2], For further analysis of the deleted Ela gene, we used the cDNA polymerase chain reaction (PCR) technique [4] Fig-1-Ela specific cDNA PCR products from Adl2wt-and CS-l-infected HeLa and Vero cells resolved on a 2% agarose gel. Ethidium bromide stain, //«elll-diEla-specific mRNAs of our mutant.…”

“…This mRNA and its pro tein have not been found in other adenoviruses, suggesting a function specific for highly on cogenic serotypes. However, this transcript and its protein are not necessary for lytic infection, as mutant CS-1 is fully viable [2] without it. Its functions like gene regulation or its role in oncogenesis have to be established.…”

A mutant of canine adenovirus type 2 with a duplication of the E1a region exhibits altered expression of early region 4

“…Adenovirus type 12 mutants have adapted to growth in a number of different cell lines (Werner & zur Hausen, 1978;Schwartz et al, 1982;Salewski et al, 1989). Most of these viruses have been shown to have terminal modifications resulting in repetitions within the ITR; others had small deletions within the E 1 a region.…”

“…0001-0002 © 1991 SGM viruses have been reported in which the alterations result in multiple reiterations within the ITRs (Schwartz et al, 1982;Larsen & Tibbetts, 1985;Sira et al, 1987;Salewski et al, 1989). The mechanisms proposed for the origin of these tandem repeats suggest that a direct repeat of part of the ITR already present at one end of the genome can go through a series of duplications as a result of (inaccurate) panhandle formation by the displaced strand during DNA replication.…”