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Journal of Virology, February 2005, p. 2287-2300, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2287-2300.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genome Packaging Sense Is Controlled by the Efficiency of the Nick Site in the Right-End Replication Origin of Parvoviruses Minute Virus of Mice and LuIII

Susan F. Cotmore1 and Peter Tattersall1,2*

Departments of Laboratory Medicine,1 Genetics, Yale University School of Medicine, New Haven, Connecticut2

Received 1 September 2004/ Accepted 1 October 2004

The parvovirus minute virus of mice (MVM) packages predominantly negative-sense single strands, while its close relative LuIII encapsidates strands of both polarities with equal efficiency. Using genomic chimeras and mutagenesis, we show that the ability to package positive strands maps not, as originally postulated, to divergent untranslated regions downstream of the capsid gene but to the viral hairpins and predominantly to the nick site of OriR, the right-end replication origin. In MVM, the sequence of this site is 5'-CTAT{blacktriangledown}TCA-3', while in LuIII a two-base insertion (underlined) changes it to 5'-CTATAT{blacktriangledown}TCA-3'. Matched LuIII genomes differing only at this position (designated LuIII and Lu{Delta}2) packaged 47 and <8% positive-sense strands, respectively. OriR sequences from these viruses were both able to support NS1-mediated nicking in vitro, but initiation efficiency was consistently two- to threefold higher for Lu{Delta}2 derivatives, suggesting that LuIII's ability to package positive strands is determined by a suboptimal right-end origin rather than by strand-specific packaging sequences. These observations support a mathematical "kinetic hairpin transfer" model, previously described by Chen and colleagues (K. C. Chen, J. J. Tyson, M. Lederman, E. R. Stout, and R. C. Bates, J. Mol. Biol. 208:283-296, 1989), that postulates that preferential excision of particular strands is solely responsible for packaging specificity. By analyzing replicative-form (RF) DNA generated in vivo during LuIII and Lu{Delta}2 infections, we extend this model, showing that positive-sense strands do accumulate in Lu{Delta}2 infections as part of duplex RF DNA, but these do not support packaging. However, replication is biphasic, so that accumulation of positive-sense strands is ultimately suppressed, probably because the onset of packaging removes newly displaced single strands from the replicating pool.

* Corresponding author. Mailing address: Department of Laboratory Medicine, 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.

Journal of Virology, February 2005, p. 2287-2300, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2287-2300.2005
Copyright © 2005, 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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