The long term objective of this work is to determine the mechanisms responsible for the transport of mRNA from the nucleus to the cytoplasm by the study of adenovirus as a model system. During an infection, transport of late viral mRNA is facilitated while transport of most cellular mRNA is impeded. This effect, which is independent of the sequence of the mRNA being transported, is due to the concerted action of the E1B-55kD (E1B) and the B4-34kD (E4) proteins. The F4 protein must associate with and properly localize a portion of the E1B protein to the sites of late viral transcription and RNA processing in the nucleus. As a working hypothesis, the effect on mRNA transport can be explained by the existence of a cellular factor required for efficient transport that is drawn to the sites of adenoviral RNA processing by the E1B/B4 complex. If this factor is sufficiently limiting, nascent cellular RNA would be deprived of the factor and no longer efficiently transported during adenoviral infection. The molecular mechanisms underlying the transport of mRNA will be determined according to two specific aims.
Aim l will identify the molecular determinants of the adenoviral proteins responsible for interacting with each other and the host cell by a mutational analysis of the key viral proteins. The significance of these determinants will be elucidated by recombining key mutations into the viral chromosome and measuring their effect on mRNA transport.
Aim 2 will identify cellular factors that specifically interact with the E1B/E4 complex. Cellular factors from uninfected cells that specifically associate with the E1B/E4 complex are candidates for the hypothesized cellular factor involved in mRNA transport. Antibodies will be generated against factors that promote the association of the viral proteins in vitro to identify and further study these cellular factors. The significance of these factors will be assessed by measuring their interaction with mutant viral proteins that are deficient in the properties characterized in Aim l. Knowledge of the functions provided by the E1B and E4 proteins in the promotion of RNA transport may permit the design of an adenovirus that lacks the E1B region but has a modified E4 region with the entire functionality required for RNA transport. Without the E1B gene, such a virus would not be fully transforming, but with the E1A gene present, could multiply to a limited extent and express high levels of a recombinant gene in the viral chromosome. This vector may provide a high level of gene expression in transient gene therapies. In addition, viruses such as HIV and HTLV must actively assist the transport of its messenger RNA out of the nucleus to reproduce. An HIV infection could be slowed or possibly stopped by disrupting this process. This research will identify the normal cellular factors that cooperate with the viral products in this process. This information could be used in the systematic development of therapeutic strategies for pathogens such as HIV whose growth depends on the modulation of mRNA transport.
