Poliovirus infection enhances the formation of two ribonucleoprotein complexes at the 3' end of viral negative-strand RNA.
To identify proteins involved in the formation of replication complexes at the 3' end of poliovirus negative-strand RNA, a combined in vitro biochemical and in vivo genetic approach was used. Five subgenomic cDNA constructs were generated to transcribe different negative-strand RNA fragments. In UV cross-linking assays, distinct differences in binding of proteins in extracts from poliovirus-infected and uninfected cells to virus-specific, radiolabeled transcripts were observed. Two proteins present in extracts from poliovirus-infected cells with approximate molecular masses of 36 and 38 kDa were shown to cross-link to the 3' end of poliovirus negative-strand RNA. Appearance of the 36- and 38-kDa proteins in UV cross-linking assays can be detected 3 to 3.5 h after infection, and cross-linking reaches maximum levels by 5 h after infection. The binding site for the 36-kDa protein overlaps with the computer-predicted loop b region of stem-loop I, the so-called cloverleaf structure, and the RNA sequence of this region is required for efficient binding. Transfection of full-length, positive-sense RNA containing a five-nucleotide substitution (positions 20 to 25) in the loop b region of stem-loop I into tissue culture cells yielded only viral isolates with a reversion at position 24 (U-->C). This finding demonstrates that the wild-type cytidine residue at position 24 is essential for virus replication. RNA binding studies with transcripts corresponding to the 3' end of negative-strand RNA suggest that complex formation with the 36-kDa protein plays an essential role during the viral life cycle.
Roehl
H H
HH
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717, USA.
Semler
B L
BL
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017955:Poliovirus / Q000235:genetics* / Q000502:physiology*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000096:biosynthesis* / Q000235:genetics; D014466:Ultraviolet Rays; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D012367:RNA, Viral; D012261:Ribonucleoproteins