Minus-strand DNA transfer, an essential step in retroviral reverse transcription, is mediated by the two repeat (R) regions in the viral genome. It is unclear whether R simply serves as a homologous sequence to mediate the strand transfer or contains specific sequences to promote strand transfer. To test the hypothesis that the molecular mechanism by which R mediates strand transfer is based on homology rather than specific sequences, we examined whether nonviral sequences can be used to facilitate minus-strand DNA transfer. The green fluorescent protein (GFP) gene was divided into GF and FP fragments, containing the 5 and 3 portions of GFP, respectively, with an overlapping F fragment (85 bp). FP and GF were inserted into the 5 and 3 long terminal repeats, respectively, of a murine leukemia virus-based vector. Utilization of the F fragment to mediate minus-strand DNA transfer should reconstitute GFP during reverse transcription. Flow cytometry analyses demonstrated that GFP was expressed in 73 to 92% of the infected cells, depending on the structure of the viral construct. This indicated that GFP was reconstituted at a high frequency; molecular characterization further confirmed the accurate reconstitution of GFP. These data indicated that nonviral sequences could be used to efficiently mediate minus-strand DNA transfer. Therefore, placement and homology, not specific sequence context, are the important elements in R for minus-strand DNA transfer. In addition, these experiments demonstrate that minus-strand DNA transfer can be used to efficiently reconstitute genes for gene therapy applications.All retroviruses replicate their genome using an RNA form to generate a DNA form in a process called reverse transcription (42). The viral RNA is characterized by short repeat (R) regions at the 5Ј and 3Ј ends (7,14). The R region at the 5Ј end is immediately followed by a unique 5Ј sequence named U5. Because the viral RNA is the mRNA or the plus strand, the first strand of DNA synthesized is complementary to the viral RNA and is referred to as the minus-strand DNA. Viral DNA synthesis initiates near the 5Ј end of the viral RNA, using a tRNA primer that binds to the primer-binding site (PBS) in the viral RNA (7). Reverse transcriptase (RT) copies R and U5 and quickly reaches the 5Ј end of the RNA template. This short stretch of DNA that contains R and U5 is referred to as minus-strand strong-stop DNA. It is thought that the RNase H activity of RT degrades the RNA template in the RNA-DNA hybrid and exposes the strong-stop DNA. The newly synthesized R in the viral DNA is complementary to the R near the 3Ј end of the viral RNA. Presumably, the complementarity facilitates alignment and hybridization of the two nucleic acids and allows RT to continue DNA synthesis, using sequences near the 3Ј end of the viral RNA as a template. This switching of the RT complex from the 5Ј end to near the 3Ј end of the viral RNA, known as minus-strand DNA transfer, is an essential step in reverse transcription (7,14). Minus-strand DNA transfer...