Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins.
This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) (wild type)] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.
Towner
J S
JS
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA.
Mazanet
M M
MM
Semler
B L
BL
D012321:DNA-Directed RNA Polymerases; D003673:Defective Viruses / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011498:Protein Precursors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D011498:Protein Precursors; D011506:Proteins; D012367:RNA, Viral; D014758:Viral Core Proteins; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases