Effects of the negative control region on expression from retroviral LTR
Authors: Lijun Wang, Dennis Haas, Stephanie Halene, Donald B Kohn
Journal: Molecular Therapy (2003)

60-bp sequence near the 5′ end of the retroviral long terminal repeat (LTR) has been called a “negative control region” (ncr) because of its demonstrated inhibitory effect on expression [1Flanagan J.R. Krieg A.M. Max E.E. Khan A.S. Negative control region at the 5′ end of the murine leukemia virus long terminal repeats.Mol. Cell. Biol. 1989; 9: 739-746Crossref PubMed Scopus (66) Google Scholar]. The ncr has been shown to have a sequence element that binds the transcriptional factor YY-1, which may act in positive or negative ways. We had shown previously that deletion of the ncr fragment from a retroviral vector (MND) led to improved expression levels in murine embryonic stem (ES) cells and hematopoietic stem cells (HSC) [2Challita P.M. Skelton D. El-Khoueiry A. Yu X.J. Weinberg K.I. Kohn D.B. Multiple modifications in cis-elements of the LTR of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma (EC) cells.J. Virol. 1995; 69: 748-755PubMed Google Scholar, 3Robbins P.B. Skelton D.M. Yu X.J. Halene S.A. Leonard E.H. Kohn D.B. Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 1998; 95: 10182-10187Crossref PubMed Scopus (124) Google Scholar]. As reported in a recent issue of Molecular Therapy, Wahlers et al. [4Wahlers A. Kustikova O. Zipfeld P.F. Itoh K. Koester M. Heberlein C. Li Z. Schiedlmeier B. Skerka C. Fehse B. Baum C. Upstream conserved sequences of mouse leukemia viruses are important for high transgene expression in lymphoid and hematopoietic cells.Mol. Ther. 2002; 6: 313-320Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar] have identified another putative transcriptional factor binding site in the ncr of the LTR, for the nuclear factor of activated T cells—NFAT. They found that this NFAT-binding site has a positive effect on expression from retroviral vector LTR, in that its deletion by site-directed mutagenesis accounted for approximately 50% of the decreased expression caused by the ncr deletion (mean fluorescence intensity of an eGFP reporter). Thus, their findings would support retaining the ncr fragment in retroviral vectors because of the positive effects on expression from the NFAT site. We have made the converse analysis of the other major motif of the ncr region, the YY-1 site. We eliminated the YY-1 binding site from the MPSV LTR by site-directed mutagenesis to a sequence that abolished binding by YY-1 as measured by electrophoretic gel shift mobility assay (EMSA) (Figs. 1A and 1B). The vector with the eliminated YY-1 site (MΔYD-eGFP) consistently showed higher expression in F9 murine embryonal carcinoma cells than did the vector with an intact MPSV LTR (MD-eGFP), directly implicating the YY-1 site in suppression of expression (Fig. 1C). MΔYD-eGFP was expressed at a slightly greater level than the MND-eGFP vector, which has the entire ncr fragment deleted, removing both the YY-1 and the putative NFAT site (Fig. 1C). The majority of the studies of Wahlers et al. were done using the LTR from the spleen focus-forming virus (SFFV), which they have previously found to be the most active for gene expression in hematopoietic cells from among a group of LTRs from different murine retroviruses, such as MOMLV or MPSV [5Baum C. Hegewisch-Becker S. Eckert H.G. Stocking C. Ostertag W. Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells.J. Virol. 1995; 69: 7541-7547Crossref PubMed Scopus (216) Google Scholar]. As they point out, the SFFV LTR is unique among the LTRs studied in that it has an adjacent Sp1 site that interacts positively with the NFAT site. The other LTRs lack this Sp1 site and therefore their NFAT sites may be less active than the one from the SFFV LTR. In F9 EC cells, vectors with site-directed deletions exclusively in the putative NFAT motif (MΔND) did show a minimal decrease in expression compared to a vector with the intact MPSV LTR (MD-eGFP) (Fig. 1C). Together, our studies and those of Wahlers et al. suggest that removal of the ncr fragment has opposing positive and negative effects on expression, from elimination of the YY-1 and NFAT sites, respectively. The way to produce a vector to capitalize on these results for maximal expression would be by elimination of the YY-1 site and retention of the NFAT site, in a vector configuration such as MΔYD-eGFP. However, it must be stated that the magnitude of the effects of the sequences from the ncr are relatively small. In murine gene transfer/BMT recipients, we compared directly expression by the MSCV and MND vectors; these vectors differ mainly by the presence of the ncr in the LTR of MSCV and its absence from MND (although MSCV also lacks one of the two LTR enhancer repeats present in MND). We could not detect significant differences in expression activity, with persistent stable expression seen from both vectors (Table 1). The in vivo studies by Wahlers et al. comparing expression by vectors with and without the NFAT site in murine gene transfer/BMT recipients also failed to achieve statistically significant differences [4Wahlers A. Kustikova O. Zipfeld P.F. Itoh K. Koester M. Heberlein C. Li Z. Schiedlmeier B. Skerka C. Fehse B. Baum C. Upstream conserved sequences of mouse leukemia viruses are important for high transgene expression in lymphoid and hematopoietic cells.Mol. Ther. 2002; 6: 313-320Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar].TABLE 1Expression by MND vs MSCV vectors 3 months after murine gene transfer/BMTaBone marrow transduction, transplantation, and analysis as in Halene et al. [7].Tissue (cell type)% Cells expressing by FACSMND-eGFP-SN (n = 4)MSCV-eGFP-SN (n = 3)BM (gran)28.024.9BM (mono)28.022.6Spleen (B)16.29.7Thymus (T)46.423.02° CFU-S79.769.5a Bone marrow transduction, transplantation, and analysis as in Halene et al. [7Halene S. Wang L. Cooper R. Bockstoce D.C. Robbins P.M. Kohn D.B. Improved expression in murine hematopoietic and lymphoid cells after transplantation of bone marrow transduced with a modified retroviral vector.Blood. 1999; 94: 3349-3357PubMed Google Scholar]. Open table in a new tab In contrast, there is another cis element of retroviruses that has a greater magnitude of influence on expression from the LTR of retroviral vectors. The “repressor binding site” (rbs), located immediately 3′ of the 5′ LTR and overlapping the primer-binding site, has been identified as a negative enhancer, suppressing expression from the adjacent LTR [6Petersen R. Kempler G. Barklis E. A stem cell-specific silencer in the primer-binding site of a retrovirus.Mol. Cell. Biol. 1991; 11: 1214-1221Crossref PubMed Scopus (76) Google Scholar]. The MOMLV rbs binds an unidentified transcriptional inhibitory factor present in murine ES cells and is the primary determinant of silencing of expression by retroviral vectors in murine ES and HSC. Replacement of the MOMLV primer-binding site region (M-eGFP) with the primer-binding site from an endogenous murine retrovirus that does not bind the inhibitory protein (MD-eGFP) is the major modification that alleviates vector silencing and results in the persistent expression seen from MSCV- and MND-based vectors in mice ([8Grez M. Akgun E. Hilnerg F. Ostertag W. Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonal stem cells.Proc. Natl. Acad. Sci. USA. 1990; 87: 9202-9206Crossref PubMed Scopus (168) Google Scholar]; D. Haas and D. B. Kohn, manuscript in preparation).