RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

Abstract

Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3 consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a proteinprotein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.Rotaviruses, the major cause of diarrhea in young animals and children, are involved in the death of more than 400,000 children under the age of five each year (40, 41). Rotaviruses are members of the Reoviridae family, and their genomes are composed of 11 segments of double-stranded RNA which encode six structural proteins and five or six nonstructural proteins. The viral replication cycle occurs entirely in the cytoplasm. Upon virus entry, the viral transcriptase synthesizes capped, but nonpolyadenylated, mRNAs (16,28). These molecules are competent to serve as templates for either translation or replication. A subset of viral mRNAs are packaged into core particles and replicated to form the genomic doublestranded RNAs of progeny virions. Morphogenesis continues and viral proteins are added to generate the middle and outer layers surrounding the core particle. Mature virions, which consist of triple-layered particles, are released from the cell by lysis (reviewed in reference 16).Cellular translation involves a myriad of proteins and elements found at both the 5Ј and 3Ј termini of the majority of cellular mRNAs in a highly regulated cascade, controlled by specific protein-RNA and protein-protein interactions. In the steps leading to translational initiation, the cap structure at the 5Ј terminus of a...

Nuclear Localization of Cytoplasmic Poly(A)-Binding Protein upon Rotavirus Infection Involves the Interaction of NSP3 with eIF4G and RoXaN

“…A fragment encoding NSP3 150 to 313 was amplified by PCR and cloned in frame with the eGFP gene into the peGFP-N1 vector (Invitrogen). Human PABP-C1 cDNA (a kind gift of T. Grange, Paris VI University, Paris, France) amplified by PCR was cloned into the pcDNA 3.1/Hygro(Ϫ) vector (Invitrogen) in frame with the Flag epitope (49). eIF4G and RoXaN cDNAs cloned into pcDNA 3.1/Hygro(Ϫ) Flag or T7tag have been described previously (7,49).…”

“…Another cellular protein, a binding partner of NSP3, has been identified and called RoXaN (49). RoXaN (ZC3H7B, FLJ13787, KIAA1031) contains several different protein-protein interaction domains: tetratricopeptide repeats (TPR) and a leucine-and aspartic acid-rich (LD) domain are present at the amino terminus, and several repeats of RNA-binding zinc fingers constitute the carboxy-terminal half.…”

The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines

“…pcDNA-T7 and pcDNA-FLAG were generated by ligation of the fragments coding for the T7 and FLAG epitope tags to the pcDNA3.1/Hygro(-) vector, respectively (Vitour et al, 2004). cDNAs of mouse coronin 2, coronin 3 and Rab5a were amplified by polymerase chain reaction (PCR) from an MIN6 cDNA library.…”

The Induction Levels of Heat Shock Protein 70 Differentiate the Vulnerabilities to Mutant Huntingtin among Neuronal Subtypes

“…The substrates of Cbl-b include the epidermal growth factor receptor, colony-stimulating factor 1 receptor, hepatocyte growth factor receptor/Met, and so on (Peschard and Park, 2003). The third gene, RoXaN, which is downregulated by mutant htt in striatal neurons, is a novel cellular protein that forms a ternary complex with the initiation factor 4G and rotavirus protein neuroendocrine-specific protein 3 (Vitour et al, 2004). However, the physiological functions of RoXaN remain mostly unknown.…”