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Journal of Virology, August 2005, p. 10032-10039, Vol. 79, No. 15
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.15.10032-10039.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

William Severson,1 Xiaolin Xu,2 Michaela Kuhn,3 Nina Senutovitch,4 Mercy Thokala,5 François Ferron,6 Sonia Longhi,6 Bruno Canard,6 and Colleen B. Jonsson7*

Department of Entomology, Plant Pathology and Weed Science, P.O. Box 30003, MSC 3BE, New Mexico State University, Las Cruces, New Mexico 88003,1 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada,2 Diplomand, Institute of Microbiology and Virology, University of Witten/Herdeck, Stockumer Str. 10, 58448 Witten, Germany,3 NIH RISE Undergraduate Program, Department of Biology, New Mexico State University, Las Cruces, New Mexico 88003,4 Graduate Program in Biochemistry, Department of Entomology, Plant Pathology and Weed Science, P.O. Box 30003, MSC 3BE, New Mexico State University, Las Cruces, New Mexico 88003,5 Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, 163 Avenue de Luminy, case 925, 13288 Marseille Cedex 09, France,6 Department of Biochemistry and Molecular Biology, 2000 9th Ave. S, Southern Research Institute, Birmingham, Alabama 352057

Received 22 July 2004/ Accepted 10 March 2005

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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, 2000 9th Avenue South, Southern Research Institute, Birmingham, AL 35205. Phone: (205) 581-2681. Fax: (205) 581-2093. E-mail: jonsson{at}sri.org.

Journal of Virology, August 2005, p. 10032-10039, Vol. 79, No. 15
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.15.10032-10039.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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