Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores.
Reconstitution of influenza virus nucleoprotein (NP)-RNA complexes was performed with segment 8 RNA, which was synthesized in vitro from cDNA, and NP purified from virions. Under optimum conditions established using a filter binding assay and a gel retardation assay, NP was found to bind any RNA longer than 15 nucleotides. NP-RNA complexes formed at 30 degrees C are more resistant to high concentrations of NaCl than those formed at 0 degrees C. Treatment of NP with N-ethylmaleimide gave no effect on its RNA binding activity, whereas treatment with alkaline phosphatase enhanced its RNA binding activity. The newly developed "reverse-printing" method of RNase V1-treated complexes revealed that reconstituted NP-RNA complexes carry RNase V1-sensitive sites as do native ribonucleoprotein (RNP) cores (RNA polymerase-NP-RNA complexes), implying that RNA-NP complexes structurally similar to native RNP cores are reconstituted from isolated components.
Yamanaka
K
K
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
Ishihama
A
A
Nagata
K
K
D000469:Alkaline Phosphatase / Q000378:metabolism; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D009698:Nucleoproteins / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000235:genetics*; D011061:Poly A; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic; D012965:Sodium Chloride / Q000494:pharmacology
D009698:Nucleoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D011061:Poly A; D012965:Sodium Chloride; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D012259:Ribonuclease, Pancreatic; D000469:Alkaline Phosphatase; D005033:Ethylmaleimide