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The replication of Semliki Forest virus requires four nonstructural proteins (nsPl 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

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Semliki Forest virus-specific polypeptide nsP2 is a nonstructural protein involved in multiple steps during viral RNA replication. It was recently shown to possess single-stranded RNA-stimulated ATPase and GTPase activities. Replacement of the highly conserved lysine (Lys-192) within the classical nucleotide-binding motif A/GXXGXGKS/T with asparagine abolished its NTP-hydrolyzing activity. Also, about half of nsP2 is transported into the nucleus during viral infection. Substitution of the second arginine in its nuclear localization signal (P648RRRV) with aspartic acid rendered nsP2 totally cytoplasmic. To assess the functional importance of these sequence motifs, the same mutations were introduced into a cDNA clone of Semliki Forest virus, from which infectious RNA can be produced in vitro. Transfection of an RNA encoding Lys-192 --> Asn mutation into BHK cells did not promote viral infection. However, revertants encoding the wild-type amino acid were obtained. Cells transfected with RNA coding for Arg-649 --> Asp mutation gave rise to infectious virus termed SFV-RDR. Indirect immunofluorescence and subcellular fractionation of SFV-RDR-infected cells confirmed the cytoplasmic localization of nsP2. Measurement of host DNA synthesis late in infection revealed that infection with the parental virus inhibited DNA synthesis to 10% of control cells. In contrast, infection with SFV-RDR led only to a partial shutoff of cellular DNA synthesis. Mice experiments indicated that the pathogenicity of SFV-RDR was attenuated.