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Journal of Virology, November 2006, p. 10829-10835, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01347-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cryoelectron Microscopy Structures of Rotavirus NSP2-NSP5 and NSP2-RNA Complexes: Implications for Genome Replication^,

Xiaofang Jiang,¹ Hariharan Jayaram,^,1 Mukesh Kumar,¹ Steven J. Ludtke,^1,2 Mary K. Estes,³ and B. V. Venkataram Prasad^1,2,3*

Verna and Marrs McLean Department of Biochemistry and Molecular Biology,¹ W. M. Keck Center for Computational Biology,² Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030³

Received 26 June 2006/ Accepted 16 August 2006

The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-Å central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP5_66-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP5_66-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP5_66-188, the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.

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* Corresponding author. Mailing address: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-5686. Fax: (713) 798-1625. E-mail: vprasad{at}bcm.tmc.edu.

Published ahead of print on 23 August 2006.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Howard Hughes Medical Institute and the Department of Biochemistry, Brandeis University, Waltham, Mass.

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Journal of Virology, November 2006, p. 10829-10835, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01347-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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• Arnoldi, F., Campagna, M., Eichwald, C., Desselberger, U., Burrone, O. R. (2007). Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2. J. Virol. 81: 2128-2137 [Abstract] [Full Text]