RNA binding of recombinant nucleocapsid proteins of hantaviruses.
Genes encoding the nucleocapsid (N) proteins of two hantaviruses, Hantaan virus strain 76-118 (HTN) and Puumala virus strain CG 18-20 (PUU), were expressed in Escherichia coli as histidine-tagged proteins. They were purified by metal-chelate affinity chromatography under native or denaturing conditions to near homogeneity. The soluble form of HTN N protein was associated with RNA of E. coli. Renatured N proteins were shown to bind in vitro transcribed RNA representing the hantaviral small genomic (S) RNA segment. RNA binding was shown by affinity to filter-immobilized N proteins and by gel mobility shift assays. Competition experiments using tRNA, poly(U) and poly(A)+ U indicated that binding of RNA by the N protein is nonspecific. However, direct binding of ds-RNA resulted in efficient formation of large complexes suggesting that double-stranded nucleic acids are bound preferentially. Carboxyterminal fragments of HTN and PUU N proteins containing about 100 amino acids of the carboxy termini retained full binding capacity indicating that RNA binding occurs via a carboxyterminal domain.
Gött
P
P
ZMBH, Heidelberg, Germany.
Stohwasser
R
R
Schnitzler
P
P
Darai
G
G
Bautz
E K
EK
D002213:Capsid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006476:Orthohantavirus / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D006639:Histidine