Upstream conserved sequences of mouse leukemia viruses are important for high transgene expression in lymphoid and hematopoietic cells.
Highly conserved enhancer sequences located in the upstream part of the long terminal repeat (LTR) of murine leukemia retroviruses (MLV) were reported to compromise viral gene expression in multipotent embryonic cells in vitro and to reduce the likelihood for maintenance of retroviral gene expression in hematopoietic cells in vivo. We show that deletion of these sequences (nucleotides +37 to +95) attenuates rather than increases the transcriptional activity of retroviral vectors in hematopoietic cells almost independently of the developmental lineage (erythroid, myeloid, or lymphoid). Expression rates of modified vectors were reduced by as much as 34-65%, although the strong enhancer array located in the direct repeat of the LTR was preserved. Sequence analysis and electrophoretic mobility shift assays revealed the presence of a highly conserved binding site for NFAT (nuclear factor of activated T cells) proteins that immediately neighbors a known binding site for the transcription factor Yin-Yang1 (YY1) [corrected]. Specific inactivation of the NFAT site reduced transgene expression in all cell types investigated and had a similar effect as the destruction of a neighboring SP1 motif. Combined destruction of individual motifs for NFAT, SP1, and E twenty-six transcription factors (ETS) resulted in a severe attenuation (by 40-60%) of the retroviral enhancer. These results provide novel clues for the manipulation of retrovirus replication and vector tropism.
Wahlers
Anke
A
Heinrich-Pette-Institute, Department of Cell and Virus Genetics, 20251 Hamburg, Germany.
Kustikova
Olga
O
Zipfel
Peter F
PF
Itoh
Katsuhiko
K
Koester
Markus
M
Heberlein
Christoph
C
Li
Zhixiong
Z
Schiedlmeier
Bernd
B
Skerka
Christine
C
Fehse
Boris
B
Baum
Christopher
C
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005347:Fibroblasts / Q000502:physiology; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D009052:Leukemia Virus, Murine; D008214:Lymphocytes / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D012190:Retroviridae / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes / Q000502:physiology*
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors