We are using self-replicating alphavirus vectors to express retroviral envelope and receptor proteins. Cells containing vectors encoding retroviral envelope fuse with cells expressing retroviral receptor and thereby spread the self-replicating vector. Similarly, cells containing vectors encoding retroviral receptor fuse with cells expressing retroviral envelope. This system models spread of an infectious agent by cell-cell contact. Cells containing these vectors also 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, and to study in vitro evolution of such vectors. We made infectious vesicles encoding a murine retroviral receptor and investigated what happened to the vector during serial passage in cells expressing the envelope. Variants arose during passage that were neutral or spread faster than the starting vector in tissue culture. One selected variant had a mutation in a region of the receptor that has been implicated in envelope-receptor interaction. We produced vectors encoding receptors with deletions to see how various portions of the receptor affect fusion function. We formed vectors encoding chimeric retroviral receptors tagged with green fluorescent protein or beta-lactamase to study intracellular transport of receptor and intercellular spread of vector. We investigated the effect of drugs that inhibit cytoskeleton elements on intracellular transport of receptor and production of infectious vesicles in this system. Using ?helper? vectors that encode structural proteins of Sindbis virus, we serendipitously found that the helper vectors were packaged in virus particles more frequently than previously appreciated. We began to investigate vectors encoding other retroviral structural proteins including HIV envelope and HIV gag. - Alphavirus, Sindbis, retrovirus, virus receptor, HIV, MLV, gfp.
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