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Journal of Virology, July 2002, p. 7209-7219, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.7209-7219.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA 3' Readthrough of Oncoretrovirus and Lentivirus: Implications for Vector Safety and Efficacy

Anne-Kathrin Zaiss, Sodany Son, and Lung-Ji Chang*

Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center and McKnight Brain Institute, University of Florida, Gainesville, Florida 32610-0266

Received 11 December 2001/ Accepted 19 April 2002

The expression of reporter genes driven by the same human elongation factor 1{alpha} (EF1{alpha}) 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 EF1{alpha} intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1{alpha} 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 EF1{alpha} 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.

* Corresponding author. Mailing address: Rm. R1-252, ARB, Dept. of Molecular Genetics & Microbiology, University of Florida, Gainesville, FL 32610-0266. Phone: (352) 392-3315. Fax: (352) 392-5914. E-mail: lchang{at}mgm.ufl.edu.

Journal of Virology, July 2002, p. 7209-7219, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.7209-7219.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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