ATPASE AND GTPASE ACTIVITIES ASSOCIATED WITH SEMLIKI FOREST VIRUS NONSTRUCTURAL PROTEIN NSP2

The replication of Semliki Forest virus requires four nonstructural pr oteins (nsP1 to nsP4), all derived from the same polyprotein. One of t hese, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein. On the basis of a mino acid sequence homologies, nsP2 was predicted to possess nucleosid e triphosphatase and RNA helicase activities. Here, we report the engi neered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system. B oth polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chro matography. The two recombinant proteins exhibited ATPase and GTPase a ctivities, which were further stimulated by the presence of single-str anded RNA. The activities were hot found in similarly prepared fractio ns from uninduced control cells or cells expressing an unrelated polyp eptide. Radiolabeled ribonucleoside triphosphates could be cross-linke d to both the full-length and the carboxy-terminally truncated nsP2 pr otein, and both polypeptides had RNA-binding capacity. We also express ed and purified an nsP2 variant which had a single amino acid substitu tion in the nucleotide-binding motif (Lys-192-->Asn). No nucleoside tr iphosphatase activity was associated with this mutant protein,