Mutational analysis of the bunyamwera orthobunyavirus nucleocapsid protein gene

Eifan, S.A. and Elliott, R.M. (2009) Mutational analysis of the bunyamwera orthobunyavirus nucleocapsid protein gene. Journal of Virology, 83(21), pp. 11307-11317. (doi: 10.1128/JVI.01460-09)

Abstract

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Elliott, Professor Richard
• Authors: Eifan, S.A., and Elliott, R.M.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Journal of Virology
• ISSN: 0022-538X
• ISSN (Online): 1098-5514