Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells

Abstract

Moloney murine leukaemia virus (M-MuLV) infection of embryonal carcinoma (EC) cells results in the integration of proviral DNA into the host cell genome, but not in virus production. One suggested explanation for the lack of viral gene expression in EC cells has been methylation of the integrated viral DNA. However, subsequent reports indicated that integration of the M-MuLV DNA occurs soon after infection, but that viral DNA methylation occurs considerably later. Nevertheless, viral gene expression is not observed even at early times. One possible explanation is that certain M-MuLV regulatory sequences do not function in EC cells. We now present evidence which supports this hypothesis.

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

“…12,15 In the Moloney murine leukemia virus (Mo-MuLV), a number of regulatory elements have been found to influence transcriptional down-regulation of viral genes from the LTR. These include the enhancer repeat unit, 16,17 the negatively acting cis element in the upstream conserved region (UCR) that binds the YY1 transcription factor, 18,19 and the primer binding site (PBS). 17,20,21 Methylation of cytosine bases in vector sequences has been associated with the observed transcriptional down-regulation.…”

Replication of enhancer-deficient amphotropic murine leukemia virus in human cells

“…The provirus MLV-(MOA) was assembled from a XbaI͞SpeI fragment from pHIT110 (14) containing a Moloney-murine sarcoma virus (MSV) 5Ј LTR with the U3 region replaced by the cytomegalovirus (CMV) immediate early promoter͞enhancer unit, a SpeI͞SalI fragment from p63-2 (15) with gag and the 5Ј part of the pol gene, a SalI͞NheI fragment with the 3Ј portion of the pol gene and the amphotropic env gene from pAMS (2), and a PCR-generated NheI͞EcoRI fragment with the MLV 3Ј LTR of p63-2. In plasmid pMLV-(MOA)⌬E the 3Ј LTR of pMLV-(MOA) was replaced with the 3Ј LTR from pMLV͞CRB⌬Mo (15).…”

“…The provirus MLV-(MOA) was assembled from a XbaI͞SpeI fragment from pHIT110 (14) containing a Moloney-murine sarcoma virus (MSV) 5Ј LTR with the U3 region replaced by the cytomegalovirus (CMV) immediate early promoter͞enhancer unit, a SpeI͞SalI fragment from p63-2 (15) with gag and the 5Ј part of the pol gene, a SalI͞NheI fragment with the 3Ј portion of the pol gene and the amphotropic env gene from pAMS (2), and a PCR-generated NheI͞EcoRI fragment with the MLV 3Ј LTR of p63-2. In plasmid pMLV-(MOA)⌬E the 3Ј LTR of pMLV-(MOA) was replaced with the 3Ј LTR from pMLV͞CRB⌬Mo (15). We have used the murine fibroblast line NIH͞3T3, the human fibrosarcoma line HT-1080 (ATCC CCL121), the human breast carcinoma-derived lines MCF-7 and MDA-MB-435S (supplied by Deutsches Krebsforschungszentrum Tumor Bank, Heidelberg), human B lymphoma lines Ramos and MHH-PREB-1 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), and the human kidney line 293.…”

“…Virions from 1-ml aliquots were pelleted by centrifugation at 75,000 rpm for 10 min at 4°C with a TLA-100.3 rotor in a TL-100 ultracentrifuge (Beckman Instruments). The pellet was resuspended in 20 l RT buffer [50 mM Tris⅐HCl, pH 8.3͞125 mM NaCl͞20 mM DTT͞0.58 mM MnCl 2 ͞0.05% Nonidet P-40͞10 M desoxyribosylthymine 5Ј-triphosphate (TTP)͞ 0.1 mCi/ml ␣-[ 35 S]TTP or ␣-[ 33 P]TTP (NEN)͞10 g/ml poly(rA)͞5 g/ml oligo(dT [12][13][14][15][16][17][18] ) and incubated for 60 min at 37°C. The reaction was terminated by addition 200 l stop solution (15 mM NaCl͞100 g/ml BSA͞0.1% tetrasodiumpyrophosphate) and 25 l of 60% trichloroacetic acid (TCA), kept on ice for 15 min, and filtered through HAWP02500 membranes (Millipore).…”

Presence of the adenovirus E1A-like activity in preimplantation stage mouse embryos.

“…More recently, the presence of cellular factors responsible for the stimulation of E2A transcription was demonstrated in EC stem cells but not in differentiated cells (10,13). Another line of evidence is that several viral genomes transcribed by the use of enhancers from polyomavirus, simian virus 40, and Moloney murine leukemia virus, all of which are repressed by the ElA gene products (2, 5), are repressed in EC stem cells but efficiently transcribed in differentiation-induced EC cells (3,6,11,16 solution (21) at 2.5 days. Blastocysts were flushed from uteri at 3.5 days.…”