JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheslock, S. R.
Right arrow Articles by Hu, W.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheslock, S. R.
Right arrow Articles by Hu, W.-S.

 Previous Article  |  Next Article 

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.


This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 2000 by the American Society for Microbiology. All rights reserved.