Recognition of eIF 4 G by Rotavirus NSP 3 Reveals a Basis for mRNA

eIF4G, which brings the 3! end of the mRNA close to the 5! cap-eIF4F complex (Tarun et al., 1997; Wells et al., 1998). mRNA circularization may explain the synergistic Caroline M. Groft1 and Stephen K. Burley1,2,3,4 1Laboratory of Molecular Biophysics 2 Howard Hughes Medical Institute effects of the cap and poly(A) tail on translation (Gallie, The Rockefeller University 1991; Tarun et al., 1997; Wakiyama et al., 2000; Searfoss 1230 York Avenue et al., 2001; reviewed in Sachs, 2000). New York, New York 10021 The importance of mRNA circularization in eukaryotes has been underscored by repeated discoveries of alternative circularization strategies used by animal and Summary plant viruses. For example, Barley Yellow Dwarf Luteovirus mRNAs contain a 3! sequence, which directly base Rotaviruses, segmented double-stranded RNA viruses, pairs with a 5! sequence, thereby circumventing the co-opt the eukaryotic translation machinery with the need for trans-factors while preserving message circuaid of nonstructural protein 3 (NSP3), a rotaviral funclarization (Guo et al., 2001). Many viral mRNA circularizational homolog of the cellular poly(A) binding protein tion mechanisms, however, conserve one or both halves (PABP). NSP3 binds to viral mRNA 3! consensus seof the eukaryotic protein bridge. For example, poliovirus, quences and circularizes mRNA via interactions with a positive single-stranded RNA virus that is not capped eIF4G. Here, we present the X-ray structure of the but is polyadenylated, circularizes its genome via interC-terminal domain of NSP3 (NSP3-C) recognizing a actions between a cellular poly(C) binding protein and fragment of eIF4GI. Homodimerization of NSP3-C PABP (Herold and Andino, 2001). In this case, 5! mRNA yields a symmetric, elongated, largely "-helical strucrecognition involves the poly(C) binding protein and a ture with two hydrophobic eIF4G binding pockets at the 5! RNA-cloverleaf in the poliovirus genome, instead of a dimer interface. Site-directed mutagenesis and isother5! cap-eIF4E complex. In contrast, rotaviruses, doublemal titration calorimetry documented that NSP3 and stranded RNA viruses, transcribe messages that are PABP use analogous eIF4G recognition strategies, capped but not polyadenylated. These viruses possess despite marked differences in tertiary structure. nonstructural protein 3 (NSP3), a functional homolog of PABP and the subject of this paper, which binds to the Introduction 3! end of the viral mRNA and also interacts with eIF4G (Piron et al., 1999). Most eukaryotic mRNAs contain a 5! 7-methyl-guanoThe rotaviruses are Reoviridae family members that sine cap structure and a 3! poly(A) tail, both of which cause severe gastroenteritis in children. Classified into serve as translational control loci. The cap structure acts one of seven groups based on antigenic specificity to as a nidus for assembly of the heteromeric complex structural proteins, rotaviruses from Groups A, B, and (eukaryotic initiation factor) eIF4F, which includes the C can infect humans, with Group A rotavirus causing cap binding protein eIF4E, a ribosome-recruiting factor the vast majority of human disease (Estes, 1996). NSP3 eIF4G, and an RNA helicase eIF4A that, in conjunction is a homodimeric RNA binding protein (Mattion et al., with accessory factor eIF4B, unwinds secondary struc1992; Piron et al., 1999) critical for translation of rotaviral tural elements in the 5! untranslated region (UTR) of mRNAs. NSP3 can be crosslinked to viral mRNAs in vivo messenger RNA (reviewed in Hershey and Merrick, (Poncet et al., 1993) and is required for their translation 2000). eIF4F and various other translation factors, such both in vitro and in vivo (Vende et al., 2000). Group A as eIF2, eIF3, eIF5, and IF2/eIF5B, cooperate to ensure NSP3 is a 36 kDa protein (Both et al., 1984) with two that protein synthesis begins at the desired initiation structurally and biochemically distinct domains. The N-tercodon. minal portion of NSP3 (NSP3-N) binds to a group-speAt the 3! end of the message, the poly(A) tail is recogcific tetra-nucleotide consensus sequence found at the nized by the poly(A ) binding protein (PABP), a protein 3! ends of rotaviral mRNAs (Poncet et al., 1994; Deo et important in many cellular processes, including mRNA al., 2002). The C-terminal domain, NSP3-C, competes stability, polyadenylation, and translation. PABP binds with PABP for the same segment of eIF4G (Imataka poly(A) RNA via four RRM-type RNA binding domains and et al., 1998; Piron et al., 1998). Addition of NSP3-C to also contains a small, C-terminal, conserved "-helical dotranslation extracts decreases translation of all eukarymain (Deo et al., 2001; Kozlov et al., 2001). Documented otic mRNAs (Vende et al., 2000). During the course of PABP-interacting partners include the poly(A) binding Group A rotavirus infection, NSP3 accumulates in higher protein interacting proteins 1 (Craig et al., 1998) and 2 order complexes with eIF4G, while PABP disappears (Khaleghpour et al., 2001), eIF4B (Bushell et al., 2001; from the translation machinery (Piron et al., 1998). EvenLe et al., 1997), and eIF4G (Tarun and Sachs, 1996; tually, NSP3 is thought to shut down host cell protein Imataka et al., 1998). The best studied of these interacproduction (Michel et al., 2000) by displacing PABP and tions involves RRM2 of PABP and the N terminus of utilizing the available pool of eIF4G for translation of rotaviral mRNAs bearing the 3! tetra-nucleotide consensus sequence. 3 Correspondence: burley@rockvax.rockefeller. edu Previous structural studies of NSP3 revealed the mo4 Present address: Structural GenomiX, Inc., 10505 Roselle Street,