Early Region 1 Transforming Functions Are Dispensable for Mammary Tumorigenesis by Human Adenovirus Type 9

Abstract

Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importance of the viral E1 region for tumorigenesis by adenoviruses, we investigated whether this viral transcription unit is also necessary for Ad9 to generate mammary tumors. The nucleotide sequence of the Ad9 E1 region indicated that the gene organization and predicted E1A and E1B polypeptides of Ad9 are closely related to those of other human adenovirus E1 regions. In addition, an Ad9 E1 region plasmid demonstrated focus-forming activity in both low-passage-number and established rat embryo fibroblasts, whereas a large deletion within either the E1A or E1B gene of this plasmid diminished transforming activity. Surprisingly, we found that introducing the same transformation-inactivating E1A and E1B deletions into Ad9 results in mutant viruses that retain the ability to elicit mammary tumors in rats. These results are novel in showing that Ad9 represents a unique oncogenic adenovirus in which the E4 region, rather than the E1 region, encodes the major oncogenic determinant in the rat.

Two Distinct Activities Contribute to the Oncogenic Potential of the Adenovirus Type 5 E4orf6 Protein

“…Although expression of the E1A and E1B oncoproteins is sufficient to oncogenically transform cells, earlier work by Shiroki et al already demonstrated that the in vitro growth properties and tumorigenicity of rat 3Y1 cells transformed by E1 gene products of highly oncogenic Ad12 are substantially enhanced by coexpression of Ad12 E4 (52). Further, it was shown that the E4 genes of nononcogenic Ad2 potentiate Ad2 E1-induced focus formation in CREF cells (42) and that the presence of E4 is not only a prerequisite but even the major determinant for the tumorigenic capacity of Ad9 (19,58). The E4 function critical for tumor induction by Ad9 was genetically mapped to E4orf1, which by itself is able to transform CREF cells in vitro (20,62).…”

Functions of the adenovirus E4 proteins and their impact on viral vectors

“…Similarly the morphology and growth of rat fibroblasts transformed by E1 from Ad2 was altered by addition of the E4 region (91). In contrast, Ad9 induces estrogendependent mammary tumors in female rats independently of the E1-region, and the major oncogenic determinant resides in the E4 region (92,93). These studies implicate the E4 region in oncogenicity and recent work has revealed roles for individual gene products.…”

“…Ad9 is distinct from the other human Ads in that it elicits only estrogen-dependent mammary tumors in animals (104). The oncogenic determinant maps solely to the E4 region and the product of E4orf1 (28,93). Expression of Ad9 E4orf1 is sufficient to induce a transformed phenotype and enhance oncogenicity in vivo (105).…”

Xenogenic Adenoviral Vectors

“…Oncogenes must also be removed from the vector genome. This may be more straightforward for the mastadenoviruses, where precedents exist from HAdV2/5 studies, but within this genus some viruses are more oncogenic than others [2,65] and some ORFs exhibit unexpected transforming properties [160][161][162]. Progress toward oncogene identification in FAdVl has also been made [73,111], but others may exist.…”