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Journal of Virology, April 2002, p. 3301-3308, Vol. 76, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.7.3301-3308.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The RNA Binding Domain of the Hantaan Virus N Protein Maps to a Central, Conserved Region

Xiaolin Xu,1 William Severson,2 Noah Villegas,2 Connie S. Schmaljohn,3 and Colleen B. Jonsson1,2*

Graduate Program in Molecular Biology,1 Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003,2 Virology Division, United States Army Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 217023

Received 25 September 2001/ Accepted 12 December 2001

The nucleocapsid (N) protein of hantaviruses encapsidates both viral genomic and antigenomic RNAs, although only the genomic viral RNA (vRNA) is packaged into virions. To define the domain within the Hantaan virus (HTNV) N protein that mediates these interactions, 14 N- and C-terminal deletion constructs were cloned into a bacterial expression vector, expressed, and purified to homogeneity. Each protein was examined for its ability to bind the HTNV S segment vRNA with filter binding and gel electrophoretic mobility shift assays. These studies mapped a minimal region within the HTNV N protein (amino acids 175 to 217) that bound vRNA. Sequence alignments made from several hantavirus N protein sequences showed that the region identified has a 58% identity and an 86% similarity among these amino acid sequences. Two peptides corresponding to amino acids 175 to 196 (N1) and 197 to 218 (N2) were synthesized. The RNA binding of each peptide was measured by filter binding and competition analysis. Three oligoribonucleotides were used to measure binding affinity and assess specificity. The N2 peptide contained the major RNA binding determinants, while the N1 peptide, when mixed with N2, contributed to the specificity of vRNA recognition.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003. Phone: (505) 646-3346. Fax: (505) 646-2649. E-mail: cjonsson{at}nmsu.edu.

Journal of Virology, April 2002, p. 3301-3308, Vol. 76, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.7.3301-3308.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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