RETROVIRUS REGULATION IN VITRO AND DURING DEVELOPMENT

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The goal of experiments in this proposal is to determine the structural and functional relationship between the avian leukosis-sarcoma virus enhancer and enhancer sequences within the LTRs of avian endogenous viruses (evs). Non-acute transforming avian leukosis viruses (ALVs) induce a high incidence of bursal lymphomas after infection of certain susceptible strains of chickens. Cellular transformation by these viruses is commonly associated with integration of proviral DNA near or within the cellular oncogene c-myc which results in increased expression of this gene under the control of promoter/enhancer sequences within the U3 region of the proviral LTR. In contrast, the avian endogenous viruses (evs) are weakly oncogenic in vivo. It has been suggested that low level oncogenicity of avian evs is due primarily to the absence of a strong enhancer in their LTRs that prevents them from efficiently mediating cis-activation events such as those seen in exogenous virus-induced tumors. Unexpectedly however, sequences have been identified in ev LTRs that are able to replace, in an orientation independent mariner, essential enhancer sequences in the exogenous virus LTR with which they share limited sequence similarity. By constructing ev-exogenous virus hybrid LTRs, the sequences within ev LTRs required for this effect are being localized. Preliminary data has also been obtained indicating that ev enhancer sequences interact with cellular proteins distinct from exogenous virus enhancer binding proteins. Thus, enhancer motifs within the ev LTR appear distinct from those in the exogenous virus LTR. The availability of hybrid LTRs that are transcribed efficiently yet contain enhancer motifs from the weakly oncogenic ev provides the opportunity to directly test the relationships between enhancer activity, the identity and function of distinct enhancer motifs, and oncogenesis. This will be investigated using a combined approach of protein binding studies and functional analyses on individual ev and exogenous virus enhancer domains and on ev-exogenous virus hybrid LTRs. Selected hybrid LTRs will then be used to generate replication competent virus in order to determine their growth rates in vitro and to generate virus stocks for future use in oncogenicity testing. The long range goal of these studies is to define and to potentially distinguish between regulatory elements that mediate enhancer activity in in vitro experiments and, in the future, those that are required for high level oncogenesis in vivo.