Mechanism for Control of Synthesis of Semliki Forest Virus 26S and 42S RNA

Abstract

When [baby hamster kidney BHK-21] cells infected with the Semliki Forest virus (SFV) mutant ts-4 were shifted to the nonpermissive temperature, synthesis of 26S RNA ceased, but synthesis of 42S RNA continued normally. These 2 single-stranded SFV RNA are synthesized in 2 types of replicative intermediate (RI), 26S RNA in RIb and 42S RNA in RIa. Cessation of 26S RNA synthesis after shift up in temperature was accompanied by loss of RIb. When infected cells were shifted back down to 27.degree. C, 26S RNA synthesis resumed, coincident with the reappearance of RIb. In both types of RI, the 42S minus-strand RNA is template for synthesis of plus-strand RNA. In pulse-chase experiments, RI labeled only in their minus-strand RNA were obtained. Thus followed the fate of RI assembled at 27.degree. C when they were shifted to 39.degree. C. After shift up to 39.degree. C, there was a quantitative conversion of RI in which 26S RNA was synthesized to RI in which 42S RNA was synthesized. This conversion of RIb to RIa was reversible, since RI in which 26S RNA was synthesized reappeared when the infected cultures were shifted back down to 27.degree. C. Associated with RIb, in which 26S RNA is synthesized, there is apparently a virus-specific protein that functions to promote initiation of 26S RNA transcription at an internal site on the 42S minus-strand RNA and to block transcription on the minus strand in this region by the SFV RNA polymerase that bound and was copying the minus-strand RNA from its 3'' end. A RNase sensitive region would thus result in the sequence adjacent to the one that was complementary to 26S RNA. This virus-specific protein is not a component of the SFV RNA polymerase that continues to transcribe 42S RNA, and it is temperature sensitive in ts-4 mutant-infected cells. When this virus-specific protein is not present on RI, the SFV polymerase transcribes the whole 42S minus-strand RNA and yields 42S plus-strand RNA.