RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria.
The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses.
Medimmune, Inc., Gaithersburg, Maryland 20878.
P
P
Warrener
J K
JK
Tamura
M S
MS
Collett
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011119:Polynucleotides / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015005:Yellow fever virus / Q000201:enzymology*
D011119:Polynucleotides; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase