The Moloney Murine Leukemia Virus Repressor Binding Site Represses Expression in Murine and Human Hematopoietic Stem Cells

Abstract

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34 ؉ CD38 ؊ hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.Retroviral vectors based upon the Moloney murine leukemia virus (MLV) have been used widely due to their high efficiency of stable gene transfer. Transcription from the MLV long terminal repeat (LTR) is severely repressed in mouse embryonal carcinoma (EC) cells (7,23,42,45) and mouse embryonal stem (ES) cells (13,21). Characteristics of MLV that have been shown to mediate poor expression in undifferentiated ES and EC cell lines include the inadequate function of the enhancer in the 5ЈLTR due to the lack of transcriptional activator binding sites (9,20,21,27,41,47), the negative control region (NCR) located in the U3 region of the LTR (1, 5, 10, 12, 47), and the repressor binding site (RBS) (2, 9).The RBS is an 18-bp DNA element located downstream of the 5ЈLTR which directly overlaps the MLV primer binding site (PBS) by 17 of its 18 bp (25). The MLV PBS functions by binding a proline tRNA molecule that primes reverse transcription. The existence of the overlapping RBS was first determined due to a spontaneously occurring single-base G-to-A mutation that occurred within the PBS sequence that allowed expression by MLV in F9 EC cells (2). This single base mutation was called the B2 mutation (2). Like the MLV PBS, the B2 PBS binds to a proline tRNA molecule, but with a single base-pair mismatch (4). Several other mutations that have been made within the MLV PBS have also been shown to relieve the r...

TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells

“…To date, most studies of PBSmediated restriction have been performed in mouse cells. However, it has been reported that PBS Pro -mediated restriction also occurs in human cells (24). Thus it is likely that other mammals will also induce embryonic silencing of other PBS-containing retroviruses.…”

“…Previous work from many laboratories has documented the presence of strong sequence-specific DNA-binding activity for the PBS Pro of MLV in lysates of embryonic cells (9,12,13,24). Similar tests for DNA-binding activity in F9 EC cell extracts, when performed using the DNA sequence of PBS Lys-1,2 , the PBS used by retroviruses such as visna, spuma, and Mason-Pfizer monkey virus (20)(21)(22), yielded a complex detected by EMSA with a shift indicative of similar size as the complex that binds to the PBS Pro sequence (9) (Fig.…”

The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses

“…However, it has been shown that repression of retroviral vectors containing the PBS-pro sequence is not restricted to ESCs and ECCs but is also evident in murine hematopoietic stem cells (Haas et al 2003) and ZFP809 expressing human embryonic kidney cells (Wolf and Goff 2009). Also, conditional KAP1 knockout in neural progenitor cells has recently been shown to result in ERV reactivation (Fasching et al 2015), indicating that ERV repression by KRAB-ZFPs and KAP1 plays a role in several somatic cell types.…”

Ectopic DNMT3L Triggers Assembly of a Repressive Complex for Retroviral Silencing in Somatic Cells

“…9E) or MEFs at lower passages, when HDAC1 is predominantly localized to the nucleus, DNMT3L does not further influence the subcellular localization of HDAC1 significantly. The fact that HDAC1 is already localized to the nucleus in ES cells may account for their inherent ability to suppress retroviral activity (49,50). DNMT3L and TRIM28 synergistically silence proviral sequences in ES cells through multiple mechanisms.…”