The long-term objective of this proposal is to understand the molecular mechanism by which adenovirus (Ad) inhibits host cell protein synthesis and establishes exclusive translation of its own late mRNAs. Studies during the project period demonstrated that Ad suppresses host cell protein synthesis by inactivation of a key translation initiation factor called cap binding protein, or eIF-4E, by preventing its phosphorylation. This factor is a component of a translation factor complex called cap binding protein complex, or eIF-4F, that acts as a cap-dependent RNA helicase which controls translation initiation in mammalian cells. Moreover, late Ad mRNAs are unique in that they require only very small amounts of this factor. The proposed studies build on these observations and are important not only because they attempt to understand the sophisticated control of translation by Ad, but also because they explore the most crucial points for regulation of protein synthesis in uninfected cells as well.
Specific Aim 1 will focus on how the late Ad 5' noncoding region (tripartite leader) promotes preferential translation of late viral mRNAs by minimizing their requirement for factor eIF-4F. Emphasis is placed on understanding how the leader facilitates non-linear ribosome initiation called shunting, the significance of shunting in late viral translation, and identification of cis and trans-acting factors important for preferential translation and eIF-4F independence of the tripartite leader.
In Aim 2, we will establish the site(s) for IF-4E phosphorylation (which is now in doubt), and based upon these data, attempt to identify the protein kinase(s) involved in phosphorylating and activating eIF-4E. Since Ad blocks all sites of eIF-4E phosphorylation, these studies should help identify the protein kinases, phosphatases or signalling events that Ad impairs to inhibit cell translation.
In Aim 3, we will investigate the mechanism by which Ad blocks phosphorylation of translation factor eIF-4E, and how this leads to inhibition of host cell translation. Studies will attempt to identify the viral factors that induce shutoff and to describe in molecular detail the mechanism by which Ad blocks eIF-4E phosphorylation. Taken together, these studies should help clarify the complicated mechanism by which Ad commandeers cellular protein synthesis.
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