Most eukaryotic mRNAs contain a 5Ј cap structure and a 3Ј poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3Ј end. We looked for cellular proteins that could interact with NSP3A, using the two-hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co-immunoprecipitation and in vitro with NSP3A produced in Escherichia coli. In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the Cterminal part of NSP3A, but not with its N-terminal part produced in E.coli. These results show that a physical link between the 5Ј and the 3Ј ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut-off of cellular protein synthesis.

In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3 end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA-and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.The vast majority of cellular mRNAs possess a 3Ј terminal poly(A) sequence. This sequence plays a major role in many aspects of cellular mRNA metabolism (11). Together with the 5Ј cap structure, poly(A) synergistically enhances the translation of mRNA (8,12). This effect is mediated by the poly(A)-binding protein (PABP) (20), which interacts with the 3Ј poly(A) and the eukaryotic initiation factor eIF4G (14,23,33). eIF4G is a scaffold protein that brings together eIF4E (capbinding protein), eIF4A (a helicase), PABP, and eIF3 (5). As a consequence of these multiple interactions, the 40S subunit of the ribosome, loaded with initiator tRNA and methionine, is brought to the 5Ј end of a circularized mRNA and starts scanning the 5Ј untranslated region (UTR) for the first initiation codon (21). The circularization of the mRNA via eIF4E-eIF4G-PABP and mRNA interactions (34) is thought to enhance the translation of the mRNA by allowing rapid reinitiation of new rounds of translation. Circularization of the mRNA seems particularly important for efficient and accurate initiation when competition exists between mRNA (27) or when the supply of ribosomes or initiation factors is limited (28).Rotaviruses are the major cause of diarrhea in young animals and children; they are involved in the death of more than 800,000 children each year worldwide (10). Rotaviruses are members of the Reoviridae family, and their genome is composed of 11 molecules of double-stranded RNA, which encode six structural proteins and five or six nonstructural proteins (6, 17). The virus replication cycle occurs entirely in the cytoplasm. Upon virus entry, the viral transcriptase synthesizes capped but nonpolyadenylated mRNAs (13). The viral mRNAs bear 5Ј and 3Ј untranslated regions (UTR) of variable length and are flanked by two different sequences common to all genes. In the group A rotaviruses, the 3Ј-end consensus sequence UGACC is highly conserved among the 11 genes.We have previously shown that rotavirus NSP3 presents several similarities to PABP; in rotavirus-infecte...

The 5 cap and 3 poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5-3 end crosstalk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.The 5Ј ends of all eukaryotic mRNAs are modified posttranscriptionally to carry a methylated cap structure, m 7 GpppN (1). Aside from roles in RNA splicing, stabilization, and transport, the cap structure significantly enhances the recruitment of the 40 S ribosomal subunit to the mRNA 5Ј end during translation initiation. The latter function requires recognition of the cap by the eukaryotic initiation factor (eIF) 1 4F.The eIF4F holoenzyme complex consists of the cap-binding protein eIF4E and an ATP-dependent RNA helicase (eIF4A) bound toward the N-and C-terminal ends, respectively, of a scaffold molecule eIF4G (for review see Ref.2). The C-terminal domain of eIF4G also interacts with eIF3, a complex that associates directly with the 40 S ribosomal subunit.

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3′ end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.