Abstract
The distinguishing feature of self-inactivating (SIN) retroviral vectors is the deletion of the enhancer/promoter sequences in the U3 region of the 3' long terminal repeat. This design is used to overcome transcriptional interference and prevent downstream transcription from the 3' long terminal repeat. SIN vectors were derived from a number of different retroviruses. Studies of SIN vectors show that extensive U3 deletions in HIV-based vectors do not alter viral titers or the in vitro and in vivo properties of the vectors. However, deletion of the U3 sequences in γ- and α-retroviruses correlates with defects in 3' RNA processing and reduces viral titers by >10-fold. Here, we studied the steps in the retroviral life cycle that are affected by the deletion of sequences in the 3' U3 region of Moloney murine leukemia virus-derived retroviral vectors. The results show that the amounts of both full-length and internal RNA transcripts of U3-minus vectors are reduced in the nuclei of transfected cells, an effect that is probably due to a general defect in 3' RNA processing. Furthermore, full-length RNA transcripts were also defective in terms of nuclear export. This defect was complemented by transferring the U3 region to another position within the retroviral vector, indicating that the U3 region contains position-independent cis-acting sequences that are required for the transport of full-length viral transcripts. The results also suggest that the leader region of Moloney murine leukemia virus contains inhibitory/regulatory sequences, which prevent export and mediate nuclear retention of full-length viral RNA.
Highlights
Self-inactivating retroviral vectors are characterized by the deletion of enhancer/promoter sequences in the U3 region
We studied the steps in the retroviral life cycle that are affected by the deletion of sequences in the 3 U3 region of Moloney murine leukemia virus-derived retroviral vectors
These results suggest that sequences in the U3 region could be functionally involved in different processes during retroviral RNA production
Summary
Self-inactivating retroviral vectors are characterized by the deletion of enhancer/promoter sequences in the U3 region. Results: The U3 region of Mo-MLV-derived vectors contains sequences necessary for the nuclear export of full-length viral transcripts. Subsequent to reverse transcription in a host target cell, the SIN deletion in the U3 region of the 3Ј LTR is transposed to the U3 region of the 5Ј LTR in the proviral DNA, thereby preventing the expression of full-length vector RNA This approach has been used to overcome transcriptional interference between the enhancers and promoters in the viral LTRs and the internal promoters. Improved SIN vectors derived from SNV were developed by replacing the U3 region in the 5Ј LTR with the cytomegalovirus immediate-early genes enhancer/promoter (CMV) These vectors produced viral titers close to those of the parental vectors, with the exception of differences in transcriptional efficiency between the CMV and SNV promoters [12]. We identified an additional defect in nuclear-cytoplasmic transport that affected only the full-length RNA transcripts in the packaging cells
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