Biomedical subjects

John T Patton

Publications and source records attributed to John T Patton.

21 records · Page 2Linked to original sources

PubMed2002

Rotavirus protein involved in genome replication and packaging exhibits a HIT-like fold.

Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae. Although the structures of rotavirus and other members of the Reoviridae have been extensively studied, little is known about the structures of virus-encoded non-structural proteins that are essential for genome replication and packaging. The non-structural protein NSP2 of rotavirus, which exhibits nucleoside triphosphatase, single-stranded RNA binding, and nucleic-acid helix-destabilizing activities, is a major component of viral replicase complexes. We present here the X-ray structure of the functional octamer of NSP2 determined to a resolution of 2.6 A. The NSP2 monomer has two distinct domains. The amino-terminal domain has a new fold. The carboxy-terminal domain resembles the ubiquitous cellular histidine triad (HIT) group of nucleotidyl hydrolases. This structural similarity suggests that the nucleotide-binding site is located inside the cleft between the two domains. Prominent grooves that run diagonally across the doughnut-shaped octamer are probable locations for RNA binding. Several RNA binding sites, resulting from the quaternary organization of NSP2 monomers, may be required for the helix destabilizing activity of NSP2 and its function during genome replication and packaging.

Amino Acid Sequence↗

PubMed2002

RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA.

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.

Acid Anhydride Hydrolases↗

PubMed2002

Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein.

Evidence that NSP2 plays a role in packaging and replication comes from studies on tsE(1400), a rotavirus mutant with a temperature-sensitive (ts) lesion in the NSP2 gene. Cells infected with tsE and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty. Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the ts phenotype of tsE. To gain insight into the effect of the mutation on the octameric structure and biochemical activities of tsE NSP2, the protein was expressed in bacteria and purified to homogeneity. Analytical ultracentrifugation showed that tsE NSP2 formed octamers which, like those formed by wild-type (wt) NSP2, undergo conformational change into more compact structures upon binding of nucleotides. However, exposure to Mg(2+) and the nonpermissive temperature caused disruption of the tsE octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with wt octamers. Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of tsE NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature. In contrast, these activities for wt NSP2 were higher at the nonpermissive temperature. Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication. The propensity of tsE NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.

Cell Line↗