Role of protein synthesis in the assembly of semliki forest virus nucleocapsid

Abstract

The structural proteins of Semliki Forest virus are translated as a 130K polyprotein from which the amino terminal capsid protein is cleaved immediately after being completed. We have followed the intracellular pathway of capsid protein from polysomes to the nucleo-capsids by pulse-chase experiments. After a 1-min pulse given in the middle of the infection cycle the newly formed capsid protein was quantitatively associated with the large ribosomal subunits in the polysomes. After a 2-min chase 40 to 60% of the capsid proteins disappeared from the polysomes, appearing simultaneously in the nucleocapsid pool. Since the released capsid protein could never be found free in the cytoplasm, we conclude that it was transferred directly from the polysomal ribosomes to the nucleocapsid pool. Those capsid proteins which were not transferred from the polysomes were released into the monosome pool after a 5-min chase. During longer chase periods the label in monosomes decreased slowly in parallel to a corresponding increase in the amount of labeled nucleocapsids, suggesting that monosome-associated capsid protein was also transferred to the nucleocapsid pool, albeit at a greatly reduced rate. The rapid “polysomal transfer” of capsid protein was more dominant late than early in the infection. Puromycin, cycloheximide, and sparsomycin, added immediately after the pulse, inhibited to a large extent the transfer of capsid protein to the nucleocapsid, showing that the “polysomal transfer” plays an important role in the assembly of the nucleocapsid. Inhibition of initiation of protein synthesis with pactamycin had no effect on the pathway of the pulse-labeled capsid protein.