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Journal of Virology, November 2009, p. 11307-11317, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01460-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the Bunyamwera Orthobunyavirus Nucleocapsid Protein Gene{triangledown} ,{dagger}

Saleh A. Eifan{ddagger} and Richard M. Elliott*

Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, Scotland, United Kingdom

Received 15 July 2009/ Accepted 19 August 2009

The bunyavirus nucleocapsid protein, N, is a multifunctional protein that encapsidates each of the three negative-sense genome segments to form ribonucleoprotein complexes that are the functional templates for viral transcription and replication. In addition, N protein molecules interact with themselves to form oligomers, with the viral L (RNA polymerase) protein, with the carboxy-terminal regions of either or both of the virion glycoproteins, and probably also with host cell proteins. Bunyamwera virus (BUNV), the prototype bunyavirus, encodes an N protein of 233 amino acids in length. To learn more about the roles of individual amino acids in the different interactions of N, we performed a wide-scale mutagenic analysis of the protein, and 110 single-point mutants were obtained. When the mutants were employed in a minireplicon assay to examine their effects on viral RNA synthesis, a wide range of activities compared to those of wild-type N protein were observed; changes at nine amino acid positions resulted in severely impaired RNA synthesis. Seventy-seven mutant clones were selected for use in the bunyavirus reverse genetics system, and 57 viable recombinant viruses were recovered. The recombinant viruses displayed a range of plaque sizes and titers in cell culture (from approximately 103 to 108 PFU/ml), and a number of viruses were shown to be temperature sensitive. Different assays were applied to determine why 20 mutant N proteins could not be recovered into infectious virus. Based on these results, a preliminary domain map of the BUNV N protein is proposed.

* Corresponding author. Mailing address: Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, Scotland, United Kingdom. Phone: 44 1334 463396. Fax: 44 1334 462595. E-mail: rme1{at}st-andrews.ac.uk

{triangledown} Published ahead of print on 26 August 2009.

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

{ddagger} Permanent address: King Saud University, College of Science, Dept. of Botany and Microbiology, Riyadh 11451, Saudi Arabia.

Journal of Virology, November 2009, p. 11307-11317, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01460-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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