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.

The entire nucleotide sequence of cloned cDNAs containing the 5'-untranslated region and gene 1 of Purdue-115 strain of transmissible gastroenteritis virus (TGEV) was determined. This completes the sequence of the TGEV genome, which is 28,579 nucleotides long. The gene 1 is composed of two large open reading frames, ORF1a and ORF1b, which contain 4017 and 2698 codons, respectively (stop excluded). A brief, three-codon-long ORF is present upstream of ORF1a. ORF1b overlaps ORF1a by 43 bases in the (-1) reading frame. In vitro experiments indicated that translation of the ORF1a/b polyprotein involves an efficient ribosomal frameshifting activity, as previously shown for other coronaviruses. Analysis of the predicted ORF1a and ORF1b translation products revealed that the putative functional domains identified in infectious bronchitis virus (IBV), mouse hepatitis virus (MHV) and human coronavirus 229E (HCV 229E) are all present in TGEV. The amino-terminal half of the ORF1a product exhibits greater divergence than the carboxyl-terminal half, including within the TGEV/HCV229E pair. The ORF1b protein is overall highly conserved among the above four coronaviruses, except a divergent region situated near the carboxy terminus.

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...

To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus. Compared with each other, the G genes of VHSV displayed a dissimilar overall genetic diversity which correlated with differences in geographical origin. The multiple sequence alignment of the complete G protein, showed that the divergent positions were not uniformly distributed along the sequence. A central region (amino acid position 245-300) accumulated

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferoninduced dsRNA-dependent activation of the protein kinase PKR.

Through its interaction with the 5= translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5=-capped and 3=-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3= GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3= end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)-and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCETo control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery. W hen delivered into or synthesized by the host cell, viral mRNAs compete with cellular mRNAs already present in the cytoplasm for access to the protein synthesis machinery. Recruitment of the 40S ribosomal subunit onto mRNA (translation initiation) is the rate-limiting and most controlled step of eukaryotic protein biosynthesis; hence, it is highly competitive for both cellular and viral mRNAs. The 5= cap and 3= poly(A) tail of most cellular mRNAs are joined by a protein complex containing the cap binding pr...