Virus Action and Replication 133 



after infection. Fogh and Stuart (64) published beautiful pictures 

 of crystalline arrays of whole virus particles in the cytoplasm of 

 several kinds of cells. These crystals first appeared five to six 

 hours after infection. 



In an effort to determine whether protein synthesis is required 

 for any steps in poliovirus multiplication prior to the foimation 

 of virus precursor molecules, experiments using inhibitors of pro- 

 tein synthesis were performed during that time period. The effect 

 of the inhibitors on whole virus multiplication and on infectious 

 RNA formation was measured (65). 



The amino acid analog" 5-fluorophenylalanine, which had 

 originally been shown by Ackerman et al. (66) to inhibit polio- 

 virus multiplication, was found to be effective at a concentration 

 of about 0.05 mM in completely preventing whole virus synthesis 

 while affecting synthesis of infectious RNA only slightly, if at all. 

 If FPA was left in a culture past the usual time for the onset of 

 maturation and then the effects of the drug were reversed by 

 addition of a large excess of L-phenylalanine, maturation and 

 virus-protein synthesis began within an hour, indicating that all 

 possible steps except the formation of viral coat protein had 

 occurred in a normal fashion in the presence of FPA. Puromycin, a 

 drug which inhibits protein synthesis specifically in several systems 

 in a rather different manner from that of FPA (67, 68, 69), was 

 found to inhibit both infectious RNA and whole virus synthesis 

 when added prior to two hours after infection. If the puromycin 

 was added at 2.5 hours, then about 15 per cent as much infectious 

 RNA was formed as in controls, without the formation of any 

 wliole virus; and if puromycin was added at three hours, the 

 cells produced 30 to 100 per cent of the normal amounts of infec- 

 tious RNA but only about 5 to 10 per cent of the normal yield 

 of whole virus. Addition of puromycin at the outset of infection 

 and removal 1.5 hours later resulted in a corresponding delay in 

 the beginning of infectious RNA synthesis. 



All these experiments taken together provide suggestive evidence, 

 but not proof, that the synthesis of some material, probably protein 

 in nature, is necessary for initiating the replication of poliovirus 

 RNA. This step in virus formation is not sufficiently complete 

 by two hours after infection to allow RNA replication. 



