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

Oncoretrovirus, but not lentivirus, displays a high transcriptional readthrough activity in the 3' long terminal repeat (LTR) (Zaiss et al. J. Virol. 76, 7209-7219, 2002). However, the U3-deleted, selfinactivating (SIN) lentiviral LTR also exhibits high transcriptional readthrough activity. Since the canonical "core" polyadenylation signal (AAUAAA) of the lentivirus is located in the R-U5 region, the above finding suggests that additional RNA termination signals must be present in the U3 region. Insertion of alternative termination signals including panhuman T cell leukemia virus type I polyadenylation signal, a 3' end small intron, and a tertiary tRNA motif into the lentiviral SIN LTR did not restore the transcriptional termination function. Functional dissection of the U3 region revealed that 70-80% of the termination signals reside in the transcriptional control region within 124 nt overlapping NFκB, Sp1 and TATA binding sites. Serial deletion analysis of the transcriptional control region indicates that the lentiviral enhancer/promoter elements are essential to the RNA termination function. These results characterize the mechanism of lentiviral transcriptional readthrough, which addresses important fundamental and practical issue of RNA readthrough influencing lentiviral gene function and vector safety. FindingsLentiviral vectors (LVs) establish long-term transgene expression in both dividing and non-dividing cells. Extensive deletion of all of the viral genes and most of the LTR elements are essential to the safety of this vector system [1][2][3]. The self-inactivating vector (SIN) with minimal sequence in the viral LTR has been an important safety improvement in the LV system [4]. However, the LV SIN LTR displays very high transcriptional readthrough (TR) activity [5], which potentially increases the risk of activating downstream cellular oncogenes. Here we examined activities of potential transcriptional termination elements in the SIN LVs and functionally dissected U3 sequence to identify key transcriptional termination signals. Insertion of alternative transcriptional termination elements in the LV SIN LTRThe RNA readthrough activity was determined using the sensitive Cre-loxP TE26 reporter cell line as previously described [5]. We transfected the readthrough reporter construct, EF-LTR-IRES-nlCre (rtCre), into TE26 cells which contain a loxP-nlacZ reporter gene whose expression closely correlates with the readthrough nlCre activity