We are using alpha virus vectors to express retroviral envelope and receptor proteins. Cells containing such vectors produce, to a limited extent, small vesicles containing viral envelope or receptor protein on the surface and vector RNA inside. These vesicles act like primitive viruses by fusing with cells bearing the reciprocal protein (envelope or receptor) and transferring vector RNA that goes through further rounds of replication. Formation of these infectious vesicles is enhanced by physically disrupting cells. We are using this system to study fusion mediated by viral envelope and receptor, to try to target retrovirus-infected cells with """"""""killer"""""""" vesicles, to immunize against retroviruses (in a murine model system), and to study in vitro evolution of such vectors. We made infectious vesicles encoding a murine retroviral receptor (CAT1) and showed that these vesicles specifically infect cells expressing a modified, fusogenic form of the retroviral envelope. Serial passage of this vector in these special cells resulted in increased infectivity; we are attempting to identify genetic changes in the vector responsible for the phenotypic change. We made vectors encoding a chimeric retroviral receptor protein tagged with green fluorescent protein and are using this vector to study intracellular transport of receptor and retrovirus fusion mechanisms.
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