Oligomerization of Uukuniemi virus nucleocapsid protein.
BACKGROUND
Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail.


RESULTS
A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact alpha-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two alpha-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays.


CONCLUSIONS
UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.
Katz
Anna
A
Department of Virology, Infection Biology Research Program, Haartman Institute, PO Box 21, University of Helsinki, Helsinki, Finland. anna.katz@helsinki.fi
Freiberg
Alexander N
AN
Backström
Vera
V
Schulz
Axel R
AR
Mateos
Angelo
A
Holm
Liisa
L
Pettersson
Ralf F
RF
Vaheri
Antti
A
Flick
Ramon
R
Plyusnin
Alexander
A
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D008958:Models, Molecular; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D018411:Spodoptera; D018066:Uukuniemi virus / Q000502:physiology*; D019065:Virus Assembly
D019590:Nucleocapsid Proteins