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 × 10, 5.5 × 10 and 3.1 × 10 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 2½ 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.


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