Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA.
Forty-four nucleotides at the 5' terminus of the genomic RNA of Sindbis virus can form a stable stem-loop structure and have been shown previously to be important for viral replication. The structure formed by the complement of this sequence at the 3' end of the minus-strand RNA has been proposed to be a promoter for RNA replication and as such might be bound in a specific fashion by proteins of either cellular or viral origin. Short oligonucleotide probes (either 62 or 132 nucleotides) representing the 3'-terminal sequence of the minus strand were prepared. When added to extracts from infected or uninfected cells, these probes were bound by cellular proteins, as evidenced by a shift in the electrophoretic mobility of the (labeled) oligonucleotide. Competition experiments confirmed the specificity of the interaction. Proteins of apparent molecular sizes 42 and 44 kDa, and to a lesser extent 52 kDa, could be cross-linked to the minus-sense probes by UV irradiation. A mutant minus-strand probe identical to the longer probe except for a single-nucleotide deletion corresponding to nucleotide 5 in the genomic RNA, which is lethal for the virus, was also found to bind the same proteins as the wild-type probe. The half-life of the mutant probe-cellular protein complex was threefold longer than that of the wild-type complex, however, indicating that the mutant probe was bound more tightly than the wild-type probe. We hypothesize that the binding of cellular factors may be transiently required for initiation of transcription of plus-strand RNA from the minus-strand template and that overly tight binding of such factors is deleterious for RNA replication.
Pardigon
N
N
Division of Biology, California Institute of Technology, Pasadena 91125.
Strauss
J H
JH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D002872:Chromosome Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012845:Sindbis Virus / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D014779:Virus Replication
D015345:Oligonucleotide Probes; D011506:Proteins; D012367:RNA, Viral