Poliovirus translation initiation: differential effects of directed and selected mutations in the 5' noncoding region of viral RNAs.
We have analyzed the translational defects of a number of mutations in the 5' noncoding region of poliovirus type 1 RNA. These mutations fall into three categories: (1) two mutations which resulted in temperature sensitive (ts) viruses, (2) the second-site mutations responsible for the reversion of the two ts viruses, and (3) mutations which were lethal to virus production. RNAs containing either of the ts mutations translated in vitro at levels significantly lower than wild-type levels. RNAs containing the respective second-site reversions had corrected these translational defects to levels corresponding to their viral growth potentials. Unlike in vitro translation of wild-type poliovirus RNA, translation of the RNAs which gave rise to ts mutant viruses was not stimulated by the addition of an S10 fraction from an uninfected HeLa cell extract to a rabbit reticulocyte lysate (RRL). In vitro translation of the mutant RNAs (corresponding to the ts viruses) in a RRL was stimulated by factors present in a ribosomal salt wash (RSW) from a HeLa extract, although the levels of stimulation were only half those seen for wild-type. These results suggest that the stimulatory factors present in the RSW have a decreased affinity for the mutant RNA templates but can, to some extent interact, with such RNAs if provided in high enough concentration. The in vitro translation of RNAs containing either of the lethal mutations was not stimulated by factors present in the S10 or the RSW. Taken together, our data suggest a correlation between the ability of a genetically altered RNA to respond to translation stimulatory factors in vitro and the ability of that mutation to be recovered in infectious virus. In addition, we have identified the in vivo-selected reversion of translational defects for two different ts viruses.
Dildine
S L
SL
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Stark
K R
KR
Haller
A A
AA
Semler
B L
BL
D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D015394:Molecular Structure; D017955:Poliovirus / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012367:RNA, Viral