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Journal of Virology, May 2000, p. 4755-4764, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Deletion of a Short, Untranslated Region Adjacent to the Polypurine Tract in Moloney Murine Leukemia Virus Leads to Formation of Aberrant 5' Plus-Strand DNA Ends In Vivo

Eran Bacharach,1,2 Jason Gonsky,1 David Lim,1 and Stephen P. Goff1,2,*

Department of Biochemistry and Molecular Biophysics1 and Howard Hughes Medical Institute,2 Columbia University College of Physicians and Surgeons, New York, New York 10032

Received 10 November 1999/Accepted 17 February 2000

Experiments were performed to determine the function of a 28-nucleotide untranslated sequence lying between the envelope gene and the polypurine tract (PPT) sequence in the Moloney murine leukemia virus (Mo-MuLV) genome. A mutant virus carrying a deletion of this sequence (Mo-MuLVDelta 28) replicated more slowly than wild-type (wt) virus and reverted by recombination with endogenous sequences during growth in NIH 3T3 cells. We show that this deletion did not affect the level of viral protein expression or genomic RNA packaging. Mo-MuLVDelta 28 served as a helper virus as efficiently as the wt virus; in contrast, a retroviral vector harboring this mutation exhibited reduced transduction efficiency, indicating that the mutation acts not in trans but in cis. Analysis of acutely infected cells revealed that reduced levels of viral DNA were generated by reverse transcription of the Mo-MuLVDelta 28 RNA as compared to the wt RNA. Analysis of DNA circle junctions revealed that plus-strand DNA of Mo-MuLVDelta 28 but not wt virus often retained the PPT and additional upstream sequences. These structures suggest that aberrant 5' ends of plus-strand DNA were generated by a failure to remove the PPT RNA primer and/or by mispriming at sites upstream of the PPT. These data demonstrate that the major role of the sequences immediately upstream of the PPT is specifying efficient and accurate plus-strand DNA synthesis.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University College of Physicians & Surgeons, New York, NY 10032. Phone: (212) 305-3794. Fax: (212) 305-8692. E-mail: goff{at}cuccfa.ccc.columbia.edu.

Journal of Virology, May 2000, p. 4755-4764, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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