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

Abstract

When cells infected with the Semliki Forest virus (SFV) mutant ts -4 were shifted to the nonpermissive temperature, synthesis of 26S RNA ceased, whereas synthesis of 42S RNA continued normally. These two single-stranded SFV RNAs are synthesized in two types of replicative intermediate (RI), 26S RNA in RI b and 42S RNA in RI a . Cessation of 26S RNA synthesis after shift up in temperature was accompanied by loss of RI b . When infected cells were shifted back down to 27°C, 26S RNA synthesis resumed, coincident with the reappearance of RI b . In both types of RI, the 42S minus-strand RNA is template for synthesis of plus-strand RNA. In pulse-chase experiments, we obtained RIs labeled only in their minus-strand RNA, and thus could follow the fate of RIs assembled at 27°C when they were shifted to 39°C. Our results show that, after shift up to 39°C, there was a quantitative conversion of RIs in which 26S RNA had been synthesized to RIs in which 42S RNA was synthesized. This conversion of RI b to RI a was reversible, since RIs in which 26S RNA was synthesized reappeared when the infected cultures were shifted back down to 27°C. We propose that, associated with RI b , in which 26S RNA is synthesized, there is 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 had bound and was copying the minus-strand RNA from its 3′ end. A ribonuclease-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 RIs, the SFV polymerase transcribes the whole 42S minus-strand RNA and yields 42S plus-strand RNA.