Oxidation-reduction sensitive interaction of a cellular 50-kDa protein with an RNA hairpin in the 5' noncoding region of the poliovirus genome.

Abstract

Genetic and biochemical analyses of the 5' noncoding region of poliovirus have indicated the importance of this region in both translation and amplification of the viral RNA. The role of the cellular machiner required for these events is just beginning to be revealed. Using an RNA gel retention assay, we have identified a cellular 50-kDa protein that forms a specific complex with a stable stemloop structure present in the viral 5' noncoding region. The formation of the RNA,-protein complex is dependent on the availability of free sulfhydryl groups in the protein. The possible involvement of this RNA-protein complex in the regulation of viral gene expression is discussed.Polioyirus, a member of the Picornaviridae family, is a plus-stranded RNA virus that multiplies in the cytoplasm of the infected human or monkey host cell. The 7500-nucleotide (nt) RNA molecule encodes a 220-kDa polyprotein that is cleaved by virally encoded proteases to yield the processed structural and nonstructural proteins (1). The 5' and 3' noncoding regions of the viral RNA contain sequences that mediate efficient translation and positive-strand and negative-strand amplification. Although certain sequence elements involved in these events have been identified by site-directed mutagenesis of an infectious cDNA clone (reviewed in ref.2), the identification of cellular and viral proteins that bind to these sequences has only recently been actively pursued. For example, a sequence element in the 747-nt 5' noncoding region from nt 320-631 has been found to bind cellular ribosomes internally, explaining the unusual cap-independent translation of the viral RNA (3). Subsequently, cellular proteins have been identified that bind to this region and are being examined for their role in mediating internal ribosome binding (4, 5).We Konarska and Sharp (9). Briefly, 32P-labeled RNA molecules (1 pmol; 108 cpm/ pug), made in vitro by T7 RNA polymerase, were incubated in 10 ,uI (total volume) ofbinding buffer into which extracts from uninfected or polioyirus-infected HeLa cells had been added. Seventy micrograms of total protein was present in each binding reaction mixture unless otherwise specified. Poly-(A,C,U) (Sigma)

Genetic and phylogenetic clustering of enteroviruses

“…Enteroviruses use internal initiation of protein synthesis and part of the 5'NCR serves as a landing pad for ribosomes (Sonenberg, 1990). Cellular proteins that are important for internal initiation of translation have been shown to bind to many stem-loops in the 5'NCR (del Angel et al, I989;Meerovitch eta]., Gebhard & Ehrenfeld, 1992;Haller& Semler, 1995) and in some cases a single nucleotide is critical for binding (Najita & Sarnow, 1990). This causes a strong selection force for the 5'NCR and evolution of this region may be more controlled by the cellular environment in which the virus multiplies than by the virus proteins.…”

Tumor promotion by hydrogen peroxide in rat liver epithelial cells

“…Changes in the redox state are known to influence the DNA-binding activity of several transcription factors, such as Oxy R (69), AP-1 (70), NF-κB (71), Egr-1 (72), Ets (73), Myb (74) and v-Rel. A redox mechanism is also known to contribute to cell cycle progression (76), hormone receptor interactions (77), bacteriophage DNA replication (78), light signal transduction (78,79), regulation of iron metabolism (80), protein-RNA and protein-DNA interactions and RNA transcription (81)(82)(83) and protein translation regulation (78,84). It is also known that ROS and H 2 O 2 can induce single-strand breaks in cellular DNA, oxidation of DNA bases, chromosomal aberrations and DNA-protein crosslinks (13,85,86).…”

Translation of Poliovirus mRNA

“…IV in fig. 1) interacts specifically with a 50-kD polypeptide, as determined by cross-linking experiments [48]. The 50-kD polypeptide formed a covalent Michael adduct between U202 in the loop structure and an essential SH group in the protein.…”