The overall objective of this research proposal is to define the roles of viral genes in controlling the development of adenoviruses in both permissive and non-permissive cells. In particular, we are interested in defining the requirements for early adenoviral gene products in both initiation and maintenance of oncogenic transformation in rodent cells. Combined genetic and molecular biological approaches are needed, and to this end we have isolated and are characterizing temperature-sensitive (ts), host-range (hr) and cold-sensitive (cs) hr mutants of type 5 adenovirus. The hr and cs hr mutants are all transformation-defective and fall into two complementation groups, with mutations of group I mapping physically in early region 1A and those of group II in early region 1B. Some of the cs mutants are cs for transformation and temperature-shift experiments with these, and with the cells transformed by them suggest that at least one and perhaps two early region 1 gene products are required to maintain at least part of the phenotype of adeno-transformed rodent cells. Some of the cs 1B mutants present a phenotype which suggests that a 1B gene product plays a vital role in the early-late switch during adenoviral infection. Our effort will be largely directed towards complete genetic and phenotypic characterization of these transformation-defective mutants and cs-transformed cells, and in the functions of the adenoviral early proteins involved. We also plan to isolate and characterise revertants and pseudorevertants of these and other mutants, and hope to use these to identify the nature of early protein interactions with other viral and cellular proteins. Finally, we propose to introduce site-directed point and deletion mutations to the E1, E2 and certain late genes as an extension of the genetic approach to understanding the functional organization of the proteins encoded by these sequences.
