Alleviation of murine leukemia virus repression in embryonic carcinoma cells by genetically engineered primer binding sites and artificial tRNA primers.
The primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNA(B2) to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNA(B2) to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNA(Lys1.2) suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS.
Modin
C
C
Department of Molecular and Structural Biology, University of Aarhus, Aarhus C, DK-8000, Denmark.
Lund
A H
AH
Schmitz
A
A
Duch
M
M
Pedersen
F S
FS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005822:Genetic Vectors; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012313:RNA; D012343:RNA, Transfer / Q000235:genetics*; D012357:RNA, Transfer, Lys / Q000235:genetics; D013724:Teratoma / Q000821:virology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D017931:DNA Primers; C034477:RNA primers; D012357:RNA, Transfer, Lys; D012313:RNA; D012343:RNA, Transfer