When creation of SIN lentiviral vectors was first reported (33
), the lack of transcripts from the LTR as assayed by Northern blot was provided as evidence of the complete abolition of LTR-mediated transcripts in these vectors. We have also observed that the level of transcription from the sinLTR is generally insufficient to be detectable by Northern analysis (data not shown). We were, however, concerned that this issue had not been investigated thoroughly enough, and that prompted us to develop more sensitive methods for detecting transcripts derived from the LTR of SIN vectors.
In this work, we have presented four independent lines of evidence that the sinLTR lentivectors retain promoter activity near the 5′ LTR which is responsible for the production of full-length vector genome transcripts competent for encapsidation and integration. First, we demonstrated that transduction of a lentiviral packaging cell line with a standard SIN lentivector results in the production of viable vector particles containing full-length SIN lentivector genomes (Fig. ). We also demonstrated that an HSV-TK gene placed downstream of the sinLTR, but upstream of the vector's internal promoter, is produced in quantities high enough to render cells containing this vector partially susceptible to GCV-induced cell death (Fig. ). Additionally, we demonstrated that SIN lentivectors containing no internal promoter express an eGFP transgene located directly downstream of the packaging signal and Rev-responsive element located at the 5′ end of the vector (Fig. ). Finally, using RT-PCR, we demonstrated that transcripts initiated at the U3-R junction of the 5′ LTR in SIN lentivectors can be readily detected (Fig. ), and using this assay, we conclude that the sinLTR/LR retains approximately 15% of the transcriptional activity of the wtLTR, depending on cell type.
After demonstrating the residual promoter activity in sinLTR vectors, we then sought to identify the elements remaining in the standard sinLTR vector constructs which are responsible for this activity. To provide some insight as to whether transcription initiation on the sinLTR was similar to that seen on the wtLTR, we rigorously analyzed the responsiveness of the sinLTR to the HIV-1 transactivator Tat. Tat transactivates the transcription of HIV-1 by binding to the TAR loop in nascent transcripts and directing the phosphorylation of the C-terminal domain of RNAP II by CDK9 which increases the processivity of the holoenzyme (17
). In 293 cells transduced with wtLTR-eGFP and sinLTR-eGFP vectors, we observed high-level and dose-dependent transactivation of the wtLTR by Tat (Fig. ). In contrast, the level of transcription from the sinLTR was unchanged, even at the highest level of transfected Tat plasmid (Fig. ). We thus conclude that the RNAP II holoenzyme that nucleates on the sinLTR is not Tat responsive.
The lack of Tat-responsiveness provides a clue that the process of transcriptional initiation on the sinLTR involves transcriptional activators which nucleate a RNAP II holoenzyme capable of only basal levels of transcription. Thus, we analyzed the potential for other elements in the LTR and LR to be involved in this basal promoter activity. An obvious candidate for involvement in promoting transcription from the sinLTR is the HIV-1 start site Inr, which has been identified to encompass the sequence −6 to +30 relative to the beginning of R at +1 (56
). Inr elements are found near the transcriptional start sites of many TATA-containing and TATA-less promoters (reference 47
and references therein). In the latter, the Inr is usually thought to serve as a nucleation point for the RNAP II holoenzyme and thus dictates the site at which transcripts begin. It is notable that HIV-1 has an Inr at the traditional transcriptional initiation site which has been shown to be involved in initiating transcription from the LTR in vivo (56
). There is evidence, however, that the HIV-1 start site Inr does not function efficiently in the absence of a TATA sequence at the normal −30 position relative to the transcriptional start site (56
). Indeed, a very high level of conservation of the core promoter TATA sequence has been noted in proviral isolates from HIV-1-infected individuals, indicating its importance to HIV-1 viability (12
). With the standard 400-bp U3 deletion used to create the sinLTR, the TATA sequence at this location is entirely removed (Fig. ). The remaining U3 sequence in the sinLTR does not fortuitously contain a TATA sequence, nor does it contain a sequence which can be predicted to function as a noncanonical TATA sequence as determined by analysis with Matinspector (39
We have now presented substantial evidence that the HIV-1 Inr is not responsible for the residual promoter activity in the sinLTR. In our promoter screen, the sinLTR U3-R-U5 segment alone had no detectable promoter activity (Fig. ). Additionally, lentivector constructs containing a deletion that removes the first six nucleotides of the HIV-1 Inr, a mutation that converts the first three nucleotides of R from GGG to TTT, and a mutant combining both of those modifications, were capable of expressing transgene at levels near that of the control sinLTR vector containing no mutations (Fig. ). From this, we conclude that transcription from the LTR in SIN vectors does not require the HIV-1 Inr sequence at the U3-R junction to be intact.
Several studies have demonstrated that factors that bind downstream of the transcription initiation site play a key role in transcriptional activation from the LTR in integrated HIV-1 proviruses. This led us to question whether the LR binding sites may be involved in basal transcription from the sinLTR. We approached this issue by first demonstrating that the LR from the sinLTR vector is indeed capable of promoting transcription (Fig. ) and that it does so in a manner which initiates transcription at or upstream of the first nucleotide of R (Fig. ). After mutating the binding sites for DBF1 and SP1, we observed a significant decrease in the transcriptional activity from the LR, both as an isolated fragment in a promoter screen (Fig. ) and as a mutation to the LR of a sinLTR vector (Fig. ). The mutations we made were previously demonstrated by Van Lint et al. to significantly eliminate binding of the factors to the mutated sites (52
). Although we could not eliminate all sources of transcriptional activation from the LR, we have demonstrated that mutation of DBF1 and SP1 sites in this region can reduce residual transcription from the sinLTR to roughly 20% of its original level. Unfortunately, mutation of the SP1 site is also accompanied by a significant (roughly 80%) drop in titer (Fig. ). The SP1 site in the LR overlaps stem-loop 1 (SL1) of the Ψ region (Fig. ). Although the SP1 site mutation we have used from Van Lint et al. (52
) was selected to avoid disruption of SL1, it is possible that other thermodynamically favorable conformations of Ψ are disrupted by the mutation, leading to a decrease in transcript packaging and thus vector titer. Indeed, it has been demonstrated that maintenance of the SL1 stem-loop is crucial to the packaging of HIV-1 (5
). Maintaining a high vector titer is of pivotal importance for the transduction of certain cell types, such as hematopoietic stem cells (20
), so the decrease in titer we have seen with the sinLTR vector bearing the SP1 mutation will limit its utility. It remains to be determined whether other mutations to the SP1 site will be superior in terms of both eliminating factor binding and maintaining packageability and infectivity of the vector genomes.
Predicted structure of Ψ stem-loops. The Mfold prediction of the most thermodynamically stable conformation of Ψ stem-loops 1 to 3 is diagrammed. The four nucleotides mutated in the SP1 site are indicated.
In conclusion, our finding that HIV-1-derived vectors containing the SIN deletion in the U3 region of the LTR are capable of expressing full-length genomic transcripts raises the possibility that sinLTR vectors may be susceptible to mobilization by wild-type HIV-1, which will be a concern when these vectors enter into clinical usage. Due to the susceptibility of only certain cell types to HIV-1 infection, many potential users of HIV-1-derived vectors need never worry about mobilization (e.g., those using lentivectors to transduce nonhematopoietic tissues). Mobilization is, however, a possibility in the case of lentivector-transduced progenitor or mature hematopoietic cells susceptible to HIV-1 infection, and this should be borne in mind until a sinLTR vector with a fully transcriptionally silent LTR can be developed. The lack of Tat-responsiveness of the sinLTR is fortuitous, since even in their present configuration, sinLTR vectors will be at an extreme disadvantage for mobilization by HIV-1. If a cell transduced with a sinLTR vector were to become infected with HIV-1, the virus will transcribe its own genome at very high levels in the presence of Tat, whereas the sinLTR vector would only be capable of expression at a basal level. This will result in HIV-1 genomic transcripts far outnumbering vector genomic transcripts, making the vector transcripts unlikely to be packaged.
Another issue to be considered on the basis of our data is that the basal transcription from the sinLTR may have some significance for other lentivector applications such as the development of transgenic animals by using lentiviral vectors carrying expression cassettes with cell type-specific internal promoters (30
). It is possible that low-level expression from the sinLTR may be adequate for the production of some transgene in a non-cell-type-specific manner. Such studies may benefit from a decrease in the basal transcription from the sinLTR achieved by mutating the LR DBF1 and SP1 sites.