Utilization of nonviral sequences for minus-strand DNA transfer and gene reconstitution during retroviral replication.
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.
Cheslock
S R
SR
Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia, 26506, USA.
Anderson
J A
JA
Hwang
C K
CK
Pathak
V K
VK
Hu
W S
WS
D016475:3T3 Cells; D000818:Animals; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic; D014779:Virus Replication
D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins