Nuclear Protein Binding To A Novel Retroviral Sequence

Previously, we reported the construction of a hybrid adeno-retroviral gene transfer vector capable of integrase-independent genomic integration (Zheng et al, Nat Biotechnol, 2000). This vector has a type 5 recombinant adenoviral backbone with specific elements of the Moloney murine leukemia virus (MoMLV) included. The MoMLV elements used are arranged in a unique configuration around the transgene cassette. Upstream of the cassette is a sequence containing part of the envelope sequence (ENV, 1.5 kb), the entire 5’ long terminal repeat (LTR; 0.57 kb), and the packaging sequence (0.63 kb). Downstream of the cassette is a small part of the ENV sequence (0.5 kb) and the intact 3’ LTR (0.57 kb). Zheng et al (2000) identified a 404 bp region of non-canonical integration breakpoints within the unique MoMLV upstream sequences. Objectives: This study focused on identifying interactions between the unique upstream sequences with mammalian nuclear proteins to understand the mechanism of integration mediated by this hybrid vector. Methods: To examine DNA-protein interactions that might facilitate integration events with this vector, we employed a strategy utilizing the polymerase chain reaction (PCR) to generate 11 overlapping fragments along the novel DNA segment. The PCR products were subjected to gel shift analyses using nuclear extracts derived from the human submandibular gland ductal cell line, HSG. HSG nuclear extracts reacted quite specifically with the PCR generated DNA fragments. Results: Based these results we were able to map protein-DNA binding across the entire upstream DNA fragment. We identified a 1.0 kb region of DNA exhibiting high-affinity binding around the ENV/5’ LTR junction. Interestingly, this region includes the previously identified 404 bp region in which integration breakpoints occurred within the MoMLV upstream sequences. Conclusions: These results are consistent with the possibility that mammalian nuclear proteins can specifically bind to this unique arrangement of retroviral elements.