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Journal of Virology, October 2000, p. 9571-9579, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication

Sara Rasmussen Cheslock,1,2 Jeffrey A. Anderson,1 Carey K. Hwang,1,2 Vinay K. Pathak,2 and Wei-Shau Hu2,*

Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia, 26506,1 and HIV Drug Resistance Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 217022

Received 23 May 2000/Accepted 24 July 2000

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.

* Corresponding author. Mailing address: Rm. 336, Bldg. 535, HIV Drug Resistance Program, National Cancer Institute, FCRDC, Frederick, MD 21702. Phone: (301) 846-1250. Fax: (301) 846-6013. E-mail: whu{at}mail.ncifcrf.gov.

Journal of Virology, October 2000, p. 9571-9579, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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