Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5' GG Sequence for RTPase Activity.
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.
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.
Liya
L
Hu
Dar-Chone
DC
Chow
John T
JT
Patton
Timothy
T
Palzkill
Mary K
MK
Estes
B V Venkataram
BV
Prasad
D001483:Base Sequence; D020134:Catalytic Domain; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D017353:Gene Deletion; D016679:Genome, Viral; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D043583:Nucleoside-Triphosphatase / Q000737:chemistry*; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012401:Rotavirus / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry*
D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus; D012313:RNA; D043583:Nucleoside-Triphosphatase