The Reovirus S4 Gene 3′ Nontranslated Region Contains a Translational Operator Sequence

All viruses must compete for cellular factors to efficiently express and replicate their genomes. Transcription and translation of the mammalian reovirus genome occurs within the cytoplasm of infected cells. The viral transcriptional machinery is contained within the viral core (5, 32); however, translation of viral mRNAs requires host cell factors and occurs in an environment replete with translationally functional cellular transcripts. Reovirus mRNAs lack polyadenylated tails, which serve to promote translation of many cellular mRNAs (10, 24, 30). Therefore, reovirus mRNAs must use alternative strategies to usurp the translational machinery. Viral sequences that enable reovirus mRNAs to achieve translational competence in a milieu of cellular mRNAs are not well understood. Mammalian reoviruses are nonenveloped, icosahedral viruses formed from two concentric protein shells, termed outer capsid and core (reviewed in reference 26). The reovirus genome consists of 10 discrete segments of double-stranded RNA. After removal of the viral outer capsid during entry of the virus into cells, the core becomes enzymatically active and catalyzes transcription of 10 species of viral mRNA (3, 20, 33). Capped, nonpolyadenylated messages are extruded from the viral core into the cytoplasm, where they are efficiently translated despite an abundance of polyadenylated cellular mRNAs. The s4 mRNA, which encodes the ς3 protein (22, 25), is the most efficiently translated viral transcript in both in vitro translation reactions and reovirus-infected cells (1, 21, 42). Specific nucleotides at the −3 and +4 positions relative to the AUG initiation codon contribute to translational efficiency of some mRNAs (19). However, for the reovirus s4 mRNA, nucleotide polymorphism at these positions does not significantly influence the amount of translation product produced (23), indicating that this region is not the primary determinant of translational efficiency. Sequence elements that influence translational efficiency of the s4 mRNA have not been defined. Sequences in the 3′ nontranslated regions (3′NTRs) of many viral and cellular transcripts have been shown to regulate translational efficiency. The poly(A) tail augments translation by enhancing mRNA stability and by facilitating ribosome reinitiation through indirect interactions with the 5′NTR via poly(A)-binding protein and translation factor elF-4G (reviewed in reference 29). Specific 3′NTR sequences upstream of the poly(A) tail that influence stability of mRNAs or interact with viral or cellular proteins to either enhance or repress protein synthesis also have been identified. In addition, the 3′NTRs of nonpolyadenylated transcripts also contain sequences that influence translation outcome for this class of mRNAs. For example, deletion of the 3′NTR of the nonpolyadenylated coat protein mRNA of alfalfa mosaic virus decreases the efficiency with which the coat protein transcript is translated when translation factors are limiting (14). This observation suggests that the 3′NTRs of nonpolyadenylated viral mRNAs facilitate efficient translation when competing with polyadenylated mRNAs for the translational machinery. The 3′NTRs of reovirus gene segments range from 35 to 80 nucleotides in length (9, 15). Analysis of sequence diversity of the reovirus S-class gene segments indicates that sequences at the 5′ and 3′ termini are highly conserved (6, 8, 13, 18). Conserved sequences at the termini of each of these gene segments are not limited to the NTRs and extend into the open reading frames. These observations suggest that there is selection pressure to maintain conservation of these sequences at the nucleotide level. Similar levels of conservation have been observed at the gene-segment termini of other double-stranded RNA viruses, and these conserved sequences have been implicated in regulation of translation and genome replication (27, 28, 38, 40). To test the hypothesis that conserved sequences in the 3′NTRs of reovirus mRNAs influence translation, we generated full-length and 3′NTR-truncated (3′Δ) s4 mRNAs and introduced these mRNAs into translation reactions using rabbit reticulocyte lysates. Our results indicate that discrete sequences within the S4 3′NTR serve to either enhance or repress translation of the s4 transcript. Additionally, we demonstrate that an RNA oligonucleotide corresponding to the S4 gene 3′NTR forms stable complexes with proteins contained in reticulocyte lysates. These findings indicate that the reovirus S4 3′NTR contains sequences that regulate translational efficiency of the s4 mRNA and suggest that cellular components interact with these regulatory sequences to influence translation.