Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities

Abstract

SUMMARYRotavirus NSP2 is an abundant nonstructural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg 2+ -dependent activities that proceed with similar reaction velocities, that require the catalytic His 225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RTPase activity of NSP2 may account for the absence of 5′-terminal γP on the (−) strands of the dsRNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

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

“…To further understand the RNA-binding characteristics of NSP2, particularly in regard to RNA nucleotides that lack the 5= GG, and to examine whether the C-terminal helix, which contains several basic residues, plays any role in RNA binding, we carried out binding experiments using SPR. Previous studies showed that mutating some of the basic residues that line the groove, particularly residues in the N-terminal domain, diminishes RNA binding (37). Electrostatic potential surface of the NSP2 octamer shows that in addition to a strong electropositive patch close to the cleft, the C-terminal helices of the 2-fold related NSP2 subunits form another strong positively charged patch at either end of the groove (Fig.…”

“…Crystallographic studies of NSP2 with ATP analogs show that nucleoside substrates bind within the cleft, and that ␥-phosphate hydrolysis, involving a phosphohistidine intermediate, is mediated by H225 (14). However, how NSP2 recognizes 5= RNA, which is shown to be a better substrate than NTPs for ␥-phosphate hydrolysis (37), how the narrow catalytic cleft accommodates the 5= nucleotides of RNA, and how, in the context of rotavirus replication, NSP2 makes sequence-specific interactions with conserved 5= GG dinucleotide remain unclear.…”

“…1). A strikingly consistent feature of the rotavirus (Ϫ)RNAs is the absence of ␥-phosphate at their 5= ends (9,20,37). This highly conserved feature suggests that removal of the ␥-phosphate has an obligatory function during virus replication.…”

“…This highly conserved feature suggests that removal of the ␥-phosphate has an obligatory function during virus replication. Among the rotavirus proteins, only NSP2 exhibits RNA 5= triphosphatase (RTPase) activity (37).…”

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

“…These include the NS3 protein of Kunjin virus, which contains signature Walker A and B motifs that play key roles in its RTPase activities (26), and the rotavirus NSP2 protein which employs a histidine triad for ␥ phosphate hydrolysis (41). Inspection of the VSV L protein sequence indicates that the sequence lacks any such motifs.…”