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Journal of Virology, March 2003, p. 3549-3556, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3549-3556.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins

Sameer P. Goregaoker1 and James N. Culver1,2*

Molecular and Cell Biology Program, University of Maryland,1 Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, Maryland 207422

Received 3 October 2002/ Accepted 11 December 2002

A protein-protein interaction within the helicase domain of the Tobacco mosaic virus (TMV) 126- and 183-kDa replicase proteins was previously implicated in virus replication (S. Goregaoker, D. Lewandowski, and J. Culver, Virology 282:320-328, 2001). To further characterize the interaction, polypeptides covering the interacting portions of the TMV helicase domain were expressed and purified. Biochemical characterizations demonstrated that the helicase domain polypeptides hydrolyzed ATP and bound both single-stranded and duplexed RNA in an ATP-controlled fashion. A TMV helicase polypeptide also was capable of unwinding duplexed RNA, confirming the predicted helicase function of the domain. Biochemically active helicase polypeptides were shown by gel filtration to form high-molecular-weight complexes. Electron microscopy studies revealed the presence of ring-like oligomers that displayed six-sided symmetry. Taken together, these data demonstrate that the TMV helicase domain interacts with itself to produce hexamer-like oligomers. Within the context of the full-length 126- and 183-kDa proteins, these findings suggest that the TMV replicase may form a similar oligomer.

* Corresponding author. Mailing address: Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742. Phone: (301) 405-2912. Fax: (301) 314-9075. E-mail: jc216{at}umail.umd.edu.

Journal of Virology, March 2003, p. 3549-3556, Vol. 77, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.6.3549-3556.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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