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Journal of Virology, August 2003, p. 8650-8660, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8650-8660.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem

Erik Burnett1,{dagger} and Peter Tattersall1,2*

Departments of Genetics,1 Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 065102

Received 20 February 2003/ Accepted 19 May 2003

The left-hand or 3'-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.

* Corresponding author. Mailing address: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Phone: (203) 785-4586. Fax (203) 688-7340. E-mail: peter.tattersall{at}yale.edu.

{dagger} Present address: Boyce Thompson Institute, Cornell University, Ithaca, NY 14853.

Journal of Virology, August 2003, p. 8650-8660, Vol. 77, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.16.8650-8660.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Burnett, E., Cotmore, S. F., Tattersall, P. (2006). Segregation of a Single Outboard Left-End Origin Is Essential for the Viability of Parvovirus Minute Virus of Mice. J. Virol. 80: 10879-10883 [Abstract] [Full Text]  


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