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RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids

Published online by Cambridge University Press:  01 February 1999

WEIRONG WANG
Affiliation:
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA
KELLY RIEDEL
Affiliation:
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA
PATRICIA LYNCH
Affiliation:
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA
CHEN-YA CHIEN
Affiliation:
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08855-1179, USA
GAETANO T. MONTELIONE
Affiliation:
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08855-1179, USA
ROBERT M. KRUG
Affiliation:
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179, USA
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Abstract

The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2′, which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target.

Type
Research Article
Copyright
1999 RNA Society

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