Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5′ GG Sequence for RTPase Activity

Abstract

Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5′ RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5′ γ-phosphate is a common feature of the negative-strand RNA [(−)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (−)RNAs (of group A rotaviruses) have a 5′ GG dinucleotide sequence. As the only rotavirus protein with 5′ RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (−)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (−)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5′ consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5′ GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains.

Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

“…The amino-terminal portions of the Orthoreovirus protein NS (16) and BTV protein NS2 (38) have also been shown to be essential for ssRNA binding. However, for rotavirus NSP2, in addition to the highly conserved residues in the NSP2 enzymatic cleft, the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2 (39). The aa 25 to 44 alanine mutant P9-1 protein has some residual RNA binding activity.…”

A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly

“…Crystallographic analyses demonstrated that the C-terminal helix (CTH) of NSP2 can adopt an open or closed conformation (16). In the closed conformation, monomers of NSP2 within the octamer have the CTH oriented in a position close to the rest of the molecule (Fig.…”

“…NSP2 of rotavirus strain SA11 was expressed and purified as previously described (16). The purified octameric NSP2 was concentrated to 5.7 mg/ml in PBS and biotinylated with a 5:1 molar ratio of EZ-link NHS-LC-LC-biotin (Thermo Scientific).…”

“…Monoclonal antibodies (MAbs) were made by immunizing rotavirus antibodynegative BALB/c mice with 5 g of full-length NSP2 protein in Freund's complete adjuvant. NSP2 was expressed in bacteria and purified as previously described (16). Mice were inoculated 4 weeks later with 2 g of purified NSP2 protein in Freund's incomplete adjuvant.…”

“…Replication intermediates with replicase activity isolated from RV-infected cells contain NSP2 (8,12,13), and silencing NSP2 using the SA11 tsE(1400) mutant results in a nearly complete loss of dsRNA synthesis at the nonpermissive temperature (14). The NSP2 octamer, formed by tail-to-tail interactions of two NSP2 tetramers, is predicted to be the functional form of the protein (6,15), and the C-terminal helical tail (CTH) has been implicated in viroplasm formation (16). In addition to its enzymatic functions, NSP2 interacts with several ligands, including NSP5, VP1, ssRNA, and tubulin (8,(17)(18)(19).…”