ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2.
The replication of Semliki Forest virus requires four nonstructural proteins (nsP1 to nsP4), all derived from the same polyprotein. One of these, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein. On the basis of amino acid sequence homologies, nsP2 was predicted to possess nucleoside triphosphatase and RNA helicase activities. Here, we report the engineered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system. Both polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chromatography. The two recombinant proteins exhibited ATPase and GTPase activities, which were further stimulated by the presence of single-stranded RNA. The activities were not found in similarly prepared fractions from uninduced control cells or cells expressing an unrelated polypeptide. Radiolabeled ribonucleoside triphosphates could be cross-linked to both the full-length and the carboxy-terminally truncated nsP2 protein, and both polypeptides had RNA-binding capacity. We also expressed and purified an nsP2 variant which had a single amino acid substitution in the nucleotide-binding motif (Lys-192-->Asn). No nucleoside triphosphatase activity was associated with this mutant protein.
Rikkonen
M
M
Institute of Biotechnology, University of Helsinki, Finland.
Peränen
J
J
Kääriäinen
L
L
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D012672:Semliki forest virus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D017931:DNA Primers; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases