Cleavage Defect in the Non-structural Polyprotein of Semliki Forest Virus has Two Separate Effects on Virus RNA Synthesis

Abstract

When Semliki forest virus ts-4 mutant infected [chicken fibroblast] cultures are grown at the permissive temperature (28.degree. C) and shifted to the restrictive temperature (39.degree. C), 2 different defects in RNA synthesis are manifested: the synthesis of 26S RNA is stopped within 60 min and the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28.degree. C, indicating that no new active RNA polymerase is formed at 39.degree. C. Accumulation of a non-structural precursor protein with an apparent MW of about 220,000 (ns220) was demonstrated in ts-4 infected cultures shifted to 39.degree. C. Ns220 was labeled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns220 are located near the initiation site at the 5''-end of the 42S RNA. The viral specificity of ns220 was shown by its disappearance after a shift to 28.degree. C and by labeling in the presence of sucrose, when no host cell protein synthesis is detectable. The 2 functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein took place at the permissive temperature, the RNA polymerase would remain active also at 39.degree. C, but the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation.