Pardigon, Nathalie and Strauss, James H. (1992) Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA. Journal of Virology, 66 (2). pp. 1007-1015. ISSN 0022-538X. http://resolver.caltech.edu/CaltechAUTHORS:PARjvir92
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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.
|Additional Information:||Copyright © 1992 by the American Society for Microbiology. Received 1 August 1991/Accepted 1 November 1991 We thank Michael Harrington for help in the quantitation of the gel retardation analyses, Michael Katze Judith Campbell, Eric Davidson, Jonathan Bradley, and Richard Kuhn for stimulating discussions, and Ellen Strauss for critical review of the manuscript. This work was supported in by grants DMB-8617372 and DMB-9104054 from the National Science Foundation, by the Pasteur Institute, Paris, France, and by a Gosney Fellowship from the California Institute of Technology.|
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|Deposited On:||26 May 2006|
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