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Semliki Forest Virus Intracellular RNA: Properties of the Multi-stranded RNA Species and Kinetics of Positive and Negative Strand Synthesis

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.

Three replicative forms of RNA (RF I, RF II, and RF III) have been isolated from BHK cells infected with Semliki Forest virus. Using analytical and rate-zonal sedimentation the mol. wt. of these replicative forms were estimated to be 8.5 x 10⁶, 5.5 x 10⁶ and 3.1 x 10⁶ respectively. After continuous labelling from 1 to 6 h post-infection, RF I constituted more than 80% of the total replicative forms. Competition hybridization experiments showed that one strand of RF I was 42S RNA which had opposite (negative) polarity to that found in the virus particle. The positive strand of RF I was 42S RNA. The negative strand of replicative intermediate (RI) was also found to be 42S RNA. No evidence was found for an RI with a 26S negative strand. RF I was shown to contain non-hydrogen bounded poly A at or near the 3' end of the component 42S positive strand. Isolation and analysis of the poly A tract from RF I on an acrylamide gel showed it to be of essentially the same average size as the poly A tract from virus particle RNA. About 30% of the RI molecules contained non-hydrogen bonded poly A. No poly U was detected in either RF I or RI. The kinetics of positive and negative strand synthesis were investigated during virus multiplication. These experiments showed that the rate of negative strand synthesis reaches a maximum 2 h post-infection and thereafter rapidly falls. The rate of positive strand synthesis increases rapidly up to 3 h post-infection and then remains constant for a further 3 to 4 h.

Received 7 February 1974; accepted 7 March 1975.