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Journal of Virology, November 2003, p. 11347-11356, Vol. 77, No. 21
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.21.11347-11356.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6

Alak Kanti Kar1 and Polly Roy1,2*

Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294,1 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom2

Received 27 May 2003/ Accepted 3 August 2003

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK110V is directly involved in ATP binding, whereas mutant R205Q in the arginine-rich motif ER205XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R205Q mutant and was also affected in the K110N mutant. Helicase activity was altered in both mutants. The mutation E157N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.

* Corresponding author. Mailing address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom. Phone: 44 0 20 7927 2324. Fax: 44 0 20 7927 2839. E-mail: polly.roy{at}lshtm.ac.uk.

Journal of Virology, November 2003, p. 11347-11356, Vol. 77, No. 21
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.21.11347-11356.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Matsuo, E., Roy, P. (2009). Bluetongue Virus VP6 Acts Early in the Replication Cycle and Can Form the Basis of Chimeric Virus Formation. J. Virol. 83: 8842-8848 [Abstract] [Full Text]  
  • Roy, P. (2008). Bluetongue virus: dissection of the polymerase complex. J. Gen. Virol. 89: 1789-1804 [Abstract] [Full Text]  
  • Nason, E. L., Rothagel, R., Mukherjee, S. K., Kar, A. K., Forzan, M., Prasad, B. V. V., Roy, P. (2004). Interactions between the Inner and Outer Capsids of Bluetongue Virus. J. Virol. 78: 8059-8067 [Abstract] [Full Text]  
  • Nibert, M. L., Kim, J. (2004). Conserved Sequence Motifs for Nucleoside Triphosphate Binding Unique to Turreted Reoviridae Members and Coltiviruses. J. Virol. 78: 5528-5530 [Full Text]  


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