Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants.

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24 degrees C and 35 degrees C.

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis.

The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well.