The long term objective of this proposal is to develop retroviral vectors which can target entry into specific cell types. Retroviruses are RNA virus which are the causative agents of a broad range of cancers and immunodeficiencies. In the replication of the virus, the RNA is converted to double-stranded DNA and integrated into the host chromosome. This property has made retroviral vectors a preferred means of introducing genes into cells. The focus of this proposal is to understand athe mechanism of entry of murine Leukemia us (MuLV) into the target cell. This basic understanding of the requirements for a productive infection is essential for the successful targeting of the virus to specific cell types and facilitates the use of retroviral vectors for gene therapy. Two retroviruses with differing host-range are used in the studies; the ecotropic Moloney MulV (M-MuLV) and the amphotropic 4070A isolate. Retroviral entry is a complex, carefully orchestrated event. This proposal dissects four steps in the entry of the virus. The initial step is the binding of the viral envelope gene products (env) to the cell receptor. On the virus are two envelope proteins, the Surface (SU) and the Transmembrane (TM) proteins. The viral host-range is determined by the N-terminal half of SU. This region of SU has been fused with two bacteriophage M13 coat proteins. The host-range determinant of the retrovirus is presented on the surface of the phage. The use of chimeric bacteriophage allows for the rapid screening and mutagenesis of the receptor binding domain. The VRB (highly variable B) region of SU functions to stabilize the receptor specific complex. This can either be through stabilizing a conformation of SU or through secondary interactions with the receptor. To address these possibilities, a mutation bordering the VRB which is temperature-sensitive in Rat cells, but not in mouse cells will be used. The rat homolog of the ecotropic receptor (the cationic amino acid transporter) has been isolated and chimeric mouse/rat receptors have ben generated. Functional interactions with the wild type and ts virus will be examined. Subsequent to receptor binding, conformation changes occur within the SU and TM. This is a dynamic process altering protein interactions. Through the use of second-site mutations, the protein-protein interactions of the extracellular domain of TM will be defined. The final step of retroviral entry involves the fusion of the viral and cellular membranes. The amphotropic and ecotropic virus differ in their pH dependence for fusion. Through the use of chimeric ecotropic/amphotropic envelope proteins, the domain of the proteins responsible for these divergent requirements will be defined.
The final aim of this proposal develops a general scheme to select for envelope proteins with altered receptor binding domains. This lead to the isolation of virus which target athe entry through novel host cell receptors.
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