The nonstructural protein Nsp1 of Semliki Forest virus has guanine-7-methyltransferase and guanylyltransferase-like activities, required in the capping of viral mRNAs. It is palmitoylated and tightly associated with the cytoplasmic surface of the plasma membrane, endosomes, and lysosomes. To localize the acylation site(s) and the putative membrane-targeting domain, a number of deletions were made in the nsp1 gene. Most deletions resulted in the expression of nonpalmitoylated, enzymatically inactive, cytoplasmic protein. Palmitate could be released from Nsp1 with neutral hydroxylamine, indicating a thioester linkage to a cysteine residue. Therefore we mutated the conserved cysteine residues of Nsp1 to alanine. Triple mutation of Cys418, Cys419, and Cys420 resulted in nonpalmitoylated Nsp1, which was enzymatically active and still associated with membranes. However, it could be released from the membranes with 1 M NaCl, whereas 50 mM sodium carbonate (pH 12) was required to release wild type Nsp1, suggesting a conversion from an integral to a peripheral membrane protein. Indirect confocal immunofluorescence microscopy showed that the nonpalmitoylated Nsp1 colocalized with the plasma membrane marker, concanavalin A. However, it was not detected in filopodia, which were heavily stained in cells expressing wild type Nsp1. These results indicate that the acylation of Nsp1 was not needed for its targeting to the plasma membrane, but it was necessary for the migration to the filopodial extensions of the plasma membrane.
This work was supported by the Academy of Finland and Biocentrum Helsinki. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.