Attenuation of poliovirus neurovirulence is the result of a limited number of nucleotide changes in the poliovirus genome. A major site for attenuation of Type 3 poliovirus (position 472) occurs in a noncoding region of the genome that is involved in the regulation of viral translation. Exactly how (and if) changes in translation result in poliovirus attenuation remain to be established. Mola, Paul, and Wimmer, Science, 254, 1647-1651(1991) have reported that an in vitro translation system derived from uninfected HeLa cells translated added poliovirus RNA in a manner that closely mimics the full viral replicative cycle in human cells. More recently, Barton and Flanegan, J. of Virology, 67, 822- 831, (1993), have provided additional information about this system. We are using this system to determine whether the genomic changes that decrease neurovirulence exhibit an effect on the in vitro translation of virion RNA. The response of HeLa cell extracts to the addition of wild-type poliovirus RNA are being evaluated and will be used to compare the response to RNA derived from the attenuated Sabin strain. The goal of this project is to obtain further information concerning the function of the nucleotide changes that result in attenuation of the poliovirus genome. Understanding the interaction of these sequences with viral and host factors could lead to further changes in the attenuated genome that would result in increased stability of the attenuated phenotype. French researchers have recently demonstrated a previously undetected and essential role of the region surrounding nucleotide 472 in viral RNA synthesis. This observation provides several possible explanations for our experimental results indicating that inhibition of poliovirus RNA synthesis seen with protein synthesis inhibitors that inhibit ribosomal activity is not seen when protein synthesis inhibitors are used that allow ribosomal activity while preventing the synthesis of functional proteins. We believe our results demonstrate the existence of a viral feedback regulatory mechanism linking poliovirus RNA synthesis to ribosomal activity. Coupled with the French results, indicating a dual role for the regulatory region surrounding nucleotide 472, these results suggest that attenuation of poliovirus neurovirulence may result from nucleotide changes that affect the viral feedback regulatory mechanism.
