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...

Rotavirus nonstructural protein NSP3 interacts specifically with the 3 end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine-and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.The cytoplasmic poly(A)-binding protein (PABP-C1) is considered a bona fide translation initiation factor which enhances translation by binding the 3Ј poly(A) tail of the cellular mRNAs and simultaneously interacting with eukaryotic translation initiation factor 4G (eIF4G) (27,29). eIF4G is a scaffold protein that allows mRNA circularization by providing sites of interaction for PABP-C1 and eIF4E, the protein that binds the 5Ј end of capped mRNAs. eIF4G then coordinates the assembly of several other translation initiation factors, such as eIF4A, eIF3, and the small ribosomal subunit (37). In synergy with the cap structure present at the 5Ј end of most mRNAs, PABP-C1 stimulates the translation of cellular polyadenylated mRNAs by enhancing 40S ribosome subunit recruitment and 60S subunit joining (20). Furthermore, PABP-C1 binding to eIF4G increases the affinity of eIF4E for the cap structure (6, 28) by lowering its dissociation rate. Thus, PABP-C1 enhances translation by promoting the binding of mRNA to eIF4G and by lowering dissociation of the 5Ј cap structure from the eIF4G/eIF4E complex. Normally evenly dispersed throughout the cytoplasm, PABP-C1 is redistributed into stress granules (SGs) under conditions of stress, such as oxidative stress or heat shock (22). SGs are cytoplasmic foci formed by the condensation of mRNAs stalled during translation and bound by the related RNA-binding proteins TIA-1 and TIA-R. SGs are not translationally competent, but rather serve as local storage and protection compartments for mRNAs under translational arrest during cellular stress. Although it is primarily cytoplasmic, PABP-C1 has been detected nevertheless in the nucleus of several mammalian cells (1, 17, 50, 51) associated with nuclear pre-mRNP (17). PABP-C1 is thus regarded as a shuttling protein that participates in mRNA ma...

Polypeptide chain initiation factor eIF4GI undergoes caspasemediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m 7 GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-MouthDisease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed. Cell Death and Differentiation (2000) 7, 628 ± 636.