RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy.
The expression of reporter genes driven by the same human elongation factor 1alpha (EF1alpha) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1alpha intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1alpha transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1alpha intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3'-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3' polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.
Zaiss
Anne-Kathrin
AK
Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center and McKnight Brain Institute, University of Florida, Gainesville, Florida 32610-0266, USA.
Son
Sodany
S
Chang
Lung-Ji
LJ
D000818:Animals; D017930:Genes, Reporter; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007438:Introns; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D026721:RNA 3' End Processing; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012449:Safety; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes
D020648:Peptide Elongation Factor 1; D012367:RNA, Viral