No abstract

We have further analyzed structure and expression of the E1 region of the transformation-defective adenovirus 12 (Ad12) host range mutant CS-1. Using cDNA polymerase chain reaction analysis, the E1a region was found to give raise to five transcripts analogous to the Ad12wt 13S, 12S, US, 10S species and the normal 9S mRNA. Due to loss of a splice acceptor site at position 852, the Ad12wt-specific 9.5S transcript cannot be synthesized by the mutant CS-1. The fact that the virus is, however, completely viable indicates that this mRNA is dispensable for lytic growth of Ad12. Besides the deletions in E1a described previously, a second deletion of 31 base pairs was found in the E1b gene. It affects the start site of the E1b-specific mRNAs and destroys or eliminates the AUG sequence for the E1b 19-kD protein. As shown earlier, normal E1b mRNA expression is found in mutant-infected cells, but no 19-kD E1b protein was immunoprecipitated by an E1-specific rat tumor serum.

ESSEN MEDICAL SCHOOL, HWELAND STR. 55,45122 ESSEN, FRG, essende ABSTRACT The transformation defective Vero-cell host range mutant CS-I of the highly omgenic denoviruS type 12 (Adl2) has a 69 bp deletion in the EIA gene that nmoves the carboxyterminal half of the conserved ngion 2 andtheaminotaminalhelfoftheM12spccific~calledspacerthatsams to play a pivotal role in the onw@nicity of the virus. Despite its Mliency evidence that the EIA 13s protein of CS-I ntaios the activity to bind the "pocket" proteins plOS', p107 as well as p130 in nuclear extract binding assays using GST-EIA-fusion proteins and western blot analysis. In comparison to wild type E1A the mutant EIA protein is able to disnqt E2F containing nuclear complexes as judged from gel-shif~ experiments with purified 12s and 13s EIA proteins, after transfection with an EIAexpression vector or infection with the respeaivc virus. Moteover, in traolsicnt cxpnssion assays the 13s EIA protein from both, Ad12 WT and CS-I mutant was shown to transactlv ' ate thc distal W r proximal promoter of Adl2, recently shown to contain cooperating E2F-1-and An-motifs or au EZF-S-Iike-motif, rcspcctively, in a comparable manuer. The same nsults wen obtained from Inusfedon assays using other typical E2F-motif containing promoters, like the Ads E2 or the human Qhydrofolatc duaase promoter. These data suggest that e&cient infection by the oncogenic Ad12 and the comlated virus-iduccd reproglammiq of the infected cells, including induction of cell cycle relevant mechaoisms (c.g. E2F-activation)