Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases.
Using an assay capable of detecting sequence-specific RNA/protein interactions in mammalian cells, we demonstrate that the poliovirus and rhinovirus 3C proteinases are able to bind structured target RNA sequences derived from their respective 5' noncoding regions in vivo. Specific RNA binding by poliovirus 3C was found to be dependent on the integrity of stem-loop d of the RNA cloverleaf structure located at the 5' end of poliovirus genomic RNA. In contrast, mutation of stem-loop b did not prevent this in vivo interaction. However, mutation of stem-loop b, which serves as the RNA binding site for a cellular co-factor important for efficient poliovirus replication, did significantly attenuate the efficiency of 3C RNA binding in vivo and 3CD RNA binding in vitro. This in vivo protein:RNA binding assay was also used to identify several residues in 3C that are critical for RNA binding, but dispensable for 3C proteinase activity. The mammalian cell-based RNA binding assay described in this study may have considerable potential utility in the future detection or analysis of in vivo RNA/protein interactions unrelated to the 3C/RNA interaction described here.
Blair
W S
WS
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Parsley
T B
TB
Bogerd
H P
HP
Towner
J S
JS
Semler
B L
BL
Cullen
B R
BR
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010849:Picornaviridae / Q000201:enzymology* / Q000235:genetics; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012229:Rhinovirus / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D015533:Transcriptional Activation; D014162:Transfection
D015696:Gene Products, tat; D012367:RNA, Viral; D015500:Chloramphenicol O-Acetyltransferase; D010450:Endopeptidases