Essential amino acids of the hantaan virus N protein in its interaction with RNA.
The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.
Severson
William
W
Department of Entomology, Plant Pathology and Weed Science, New Mexico State University, Las Cruces, 88003, USA. venue
Xu
Xiaolin
X
Kuhn
Michaela
M
Senutovitch
Nina
N
Thokala
Mercy
M
Ferron
François
F
Longhi
Sonia
S
Canard
Bruno
B
Jonsson
Colleen B
CB
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D018063:Hantaan virus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism*; D016415:Sequence Alignment; D014758:Viral Core Proteins / Q000737:chemistry / Q000378:metabolism*
D036022:Capsid Proteins; D010455:Peptides; D012367:RNA, Viral; D014758:Viral Core Proteins; C111782:nucleocapsid protein, Hantaan virus