The experiments described in this proposal are concerned with characterization of the structure and function of the nucleoproteins within which the adenovirus type 2 (Ad2) genome resides in virions and within infected cells. Several lines of evidence, including the observation that only viral DNA molecules that have replicated can support transcription of promoter-distal sequences of the Ad2 major late transcription unit, suggest that changes in the structure of these nucleoproteins might contribute to regulation of viral gene expression during productive infection. The major DNA binding domain of protein VII, the major virion DNA binding protein of the virion, will be identified by an extension of UV-crosslinking methods we have recently developed. Complementary information about protein VII-DNA interactions in the virion core nucleoprotein will be obtained using enzymatic and chemical probes to analyse, and identify, viral sequences that interact with protein VII packaging units. Antibodies will be raised to a second, very basic core protein, Mu (Mr 3-6,000) so that its role in DNA packaging may be examined. We have recently shown that core protein VII remains associated with intranuclear viral DNA throughout the early phase of infection and believe that nucleoproteins comprising protein VII and viral DNA include the templates for viral early gene expression. To test this conclusion rigorously, crosslinking experiments will be performed to determine whether protein VII and RNA polymerase II are associated with the same viral DNA molecules. Both crosslinking methods and enzymatic and chemical probes of nucleoprotein structure will also be used to investigate the composition and organization of viral nucleoproteins containing replicated viral DNA. Such methods will be used in conjunction with procedures to distinguish the various functional classes of viral DNA present during the late phase of infection, focusing particularly on nucleoproteins containing transcriptionally active DNA. The construction of viruses carrying specific mutations in the proteins VII and Mu genes is also described so that the biochemical studies summarised in the previous paragraph can be complemented by genetic approaches.
| Anderson, C W; Young, M E; Flint, S J (1989) Characterization of the adenovirus 2 virion protein, mu. Virology 172:506-12 |
