DEFECTIVE RNA REPLICATION BY POLIOVIRUS MUTANTS DEFICIENT IN 2A PROTEASE CLEAVAGE ACTIVITY

2A protease (2A(pro)) catalyzes the initial cleavage of the poliovirus polyprotein which separates the P1 structural protein precursor from the P2-P3 nonstructural protein precursor. In addition, 2A(pro) indire ctly induces cleavage of the p220 component of eukaryotic initiation f actor 4F, which is thought to contribute to the specific inhibition of host cell protein synthesis observed in virus-infected HeLa cells. Ho wever, it is unclear whether the trans function of 2A(pro) which induc es host cell shutoff is essential or merely facilitates efficient poli ovirus replication. In this study, three point mutations in 2A(pro) (D 38E, Y88L, and Y89L [S. P. Yu and R. E. Lloyd, Virology 182:615-625, 1 991]) which cause specific loss of trans but not cis cleavage function were independently introduced into the full-length poliovirus cDNA. I n addition, mutations which caused only partial loss of both cis and t rans cleavage activities (Y88S) or resulted in a wild-type phenotype ( Y88F) were individually introduced. When each of these mutant poliovir us cDNAs was transcribed and translated in vitro, normal proteolytic p rocessing of the viral polyprotein was observed, and p220 was not clea ved in those reactions containing proteases defective in trans functio n, as expected. Surprisingly, Northern (RNA) blot analysis and reverse transcriptase-PCRs performed after transfection of COS-7 or HeLa cell s with these viral RNAs revealed that Y88S and Y88L RNAs replicated at only very low levels. RNA replication could not be detected at all in cells transfected with D38E and Y89L RNAs. Taken together, the result s suggest a correlation between the function of 2A(pro) and productive poliovirus RNA replication in vivo that may be independent of the abi lity to cause p220 cleavage.