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 degrees 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 degrees C when they were shifted to 39 degrees C. Our results show that, after shift up to 39 degrees 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 degrees 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.