Transfer of primer binding site-mutated simian immunodeficiency virus vectors by genetically engineered artificial and hybrid tRNA-like primers.
Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA or tRNA. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (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 functional 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 hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA(Pro) backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.
Hansen
A C
AC
Department of Molecular and Structural Biology, Aarhus University, DK-8000 Aarhus, Denmark.
Grunwald
T
T
Lund
A H
AH
Schmitz
A
A
Duch
M
M
Uberla
K
K
Pedersen
F S
FS
D000818:Animals; D001665:Binding Sites; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012313:RNA; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D012367:RNA, Viral; D015302:Simian Immunodeficiency Virus / Q000235:genetics*; D014158:Transcription, Genetic
C034477:RNA primers; D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D012313:RNA