RESUMO
A specificity protein 1 (Sp1) zinc finger domain containing two tandem zinc fingers was fused to the C terminus of the integrase (IN) protein of the Moloney murine leukemia virus (MuLV). The integrity of the MuLV IN was completely preserved, since the fusion was conducted at the last amino acid residue of the protein. The vector pMIN-Sp1, which carried the fused MuLV IN-Sp1 zinc finger domain gene, was cotransfected with a wild-type MuLV vector pMLV-K to NIH/3T3 cells. A nonradioactive reverse transcriptase assay was performed on culture supernatants collected from the cotransfected cells to confirm the production of recombinant viruses. The expression of the fusion protein and the integration of the MuLV genome by the fusion protein were confirmed by a Northern and then a Southern hybridization analysis on the total RNA or genomic DNA extracted from cells infected by viruses collected from the supernatants of the cotransfected cells. Regions of the host chromosome that were selected by the fusion protein as the integration targets were sequenced using the TOPO(TM) cloning method on a series of PCR products generated with a nested set of primers. The percentage of positive clones screened that contained the DNA-binding sequence of the fused Sp1 zinc finger domain was around 13% (5 out of 39 clones). It was found that the Sp1 DNA-binding sequence was only present in regions that were proximal to one of the long terminal repeats of the integrated viral genome, suggesting that the fusion protein could select a target sequence for integration. The host flanking sequences determined for all the positive clones were also used as queries to perform a BLAST search on the GenBank mouse EST entries. Although matching scores for sequences of some of the clones computed were more significant than others, it was difficult to judge whether or not the integration in these clones had been targeted to some gene sequences. Most of the integration sites might exist in the introns, since we found that the probability of the gene sequences containing an Sp1 DNA-binding site was low.
