Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers

Simian immunodeficiency viruses (SN) harbor primer binding sites (PBS) matc hing tRNA(3)(Lys) or tRNA(5)(Lys). To study determinants of primer usage in SN, a SIVmac239-based vector was impaired by mutating the PBS to a sequenc e (PBS-X2) with no match to any tRNA, By cotransfection of a synthetic gene encoding a tRNA(Pro)-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild type vector. A vector with a PBS matching tRNA(Pro) was fu nctional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA(Pro)-tRNA(3)(Lys) hybrid with a match to PBS-Pro. The im portance of tRNA backbone identity was further analyzed by complementing th e PBS-X2 vector with a gene for a matching x2 primer with a tRNA(3)(Lys) ba ckbone, which led to three- to fourfold-higher titers than those observed f or the x2 primer with the tRNA(Pro) backbone. In summary, our results demon strate flexibility in PBS and primer usage for SIVmac239, with PBS-primer c omplementarity being the major determinant, in analogy with previous findin gs for murine leukemia viruses and human immunodeficiency virus type 1.