SV40 large T antigen up-regulates the retrotransposition frequency of viral-like 30 elements.
The regulation of non-autonomous retrotransposition is not known. A recombinant bearing a hygromycin gene and a viral-like 30 (VL30) retrotransposon tagged with an enhanced green fluorescent protein (EGFP) gene-based retrotransposition cassette was constructed and used for detection of retrotransposition events. Transfection of this recombinant produced retrotransposition events, detected both by EGFP fluorescence and PCR analysis, in hygromycin-selected clones of two established simian virus 40 (SV40)-transformed mouse NIH3T3 cell lines but not in normal NIH3T3 cells. The retrotransposition potential of this recombinant, as a provirus, was studied in stably transfected NIH3T3 clones. Transfection of these clones with either a wild-type or a mutant LE1135T SV40 large T antigen gene, not expressing small t protein, induced retrotransposition events at high frequencies as measured by fluorescence-activated cell scanning (FACS). In addition, measuring retrotransposition frequencies over a period of nine days following infection with isolated SV40 particles, revealed that the frequency of retrotransposition was time-dependent and induced as early as 24 h, increasing exponentially to high levels (>10(-2) events per cell per generation) up to nine days post-infection. Furthermore, ectopic expression of a cloned MoMLV-reverse transcriptase gene also produced retrotransposition events and suggested that the large T antigen most likely acted through induction of expression of endogenous reverse transcriptase genes. Our results show a direct correlation between SV40-cell transformation and VL30 retrotransposition and provide for the first time strong evidence that SV40 large T antigen up-regulates the retrotransposition of VL30 elements.
Noutsopoulos
Dimitrios
D
Laboratory of General Biology, Medical School, University of Ioannina, 45 110 Ioannina, Greece.
Vartholomatos
Georgios
G
Kolaitis
Nicolaos
N
Tzavaras
Theodore
T
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000502:physiology*; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000502:physiology; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006921:Hygromycin B / Q000378:metabolism; D051379:Mice; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009154:Mutation; D041681:NIH 3T3 Cells; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D018626:Retroelements / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D018626:Retroelements; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; D012194:RNA-Directed DNA Polymerase