Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells.
Since the integration of viral DNA in the host genome is an essential step in the replication cycle of HIV-1, an active search for inhibitors of the integration step is ongoing. Our laboratory has been working on the development of a cellular integration system. Such a system would be helpful in the study of the HIV-1 integration process and, eventually, could be used in the search for new inhibitors that selectively interfere with HIV integration. We have previously selected stable cell lines (293T-INS) that constitutively express high levels of HIV-1 integrase (IN) from a synthetic gene [FASEB J. 14 (2000) 1389]. We have now constructed linear DNA substrates containing the terminal HIV LTR sequences (so called 'mini-HIV') and EGFP as reporter gene to evaluate whether IN can improve the integration of transfected linear DNA. After electroporation of this mini-HIV we observed a 2- to 3-fold increase in EGFP expression in IN expressing cell lines relative to control cells. The increase in EGFP expression was still evident after passaging of the cells. The effect was observed with linear DNA but not with circular DNA, thus excluding an effect on DNA uptake. The increase was the highest in the 293T-INS(D64V) cell line due to an increase in the amount of total mini-HIV DNA and 2-LTR circles as quantified by Q-PCR. Our data suggest that IN over-expressed in our cell lines interacts with the incoming DNA, protects it from nuclease degradation but does not catalyze the integration as such.
Van Maele
Bénédicte
B
Laboratory of Molecular Virology and Gene Therapy, KULAK and KULeuven, Flanders, Belgium.
Van Eylen
Leen
L
Pluymers
Wim
W
Debyser
Zeger
Z
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D019427:HIV Integrase / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D014162:Transfection
D004270:DNA, Circular; D004279:DNA, Viral; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D019427:HIV Integrase