Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein.

The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3'-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus.

Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.

Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms.

Simian rotaviruses possess divergent gene constellations that originated from interspecies transmission and reassortment.

Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basic research and vaccine development. To explore the origins of simian RVs, the complete genome sequences of strains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed the genotype constellations of each virus to be determined and the phylogenetic relationships of the simian strains with each other and with nonsimian RVs to be elucidated.

Group A human rotavirus genomics: evidence that gene constellations are influenced by viral protein interactions.

Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references.

Non-structural protein NSP2 induces heterotypic antibody responses during primary rotavirus infection and reinfection in children.

Rotaviruses are the single most important causes of severe acute diarrhoea in children worldwide. Despite success in developing vaccines, there is still a lack of knowledge about many components of the immune response, particularly those to non-structural proteins. This study established radioimmunoprecipitation (RIP) assays using labeled G1P[8], G2P[4], and G4P[6] human rotaviruses to examine the spectrum and duration of rotavirus antibodies in sera collected sequentially for 18-36 months from 27 children after hospitalization for primary rotavirus gastroenteritis.

Crystallographic and biochemical analysis of rotavirus NSP2 with nucleotides reveals a nucleoside diphosphate kinase-like activity.

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation.

Coupling of rotavirus genome replication and capsid assembly.

The Reoviridae family represents a diverse collection of viruses with segmented double-stranded (ds)RNA genomes, including some that are significant causes of disease in humans, livestock, and plants. The genome segments of these viruses are never detected free in the infected cell but are transcribed and replicated within viral cores by RNA-dependent RNA polymerase (RdRP). Insight into the replication mechanism has been provided from studies on Rotavirus, a member of the Reoviridae whose RdRP can specifically recognize viral plus (+) strand RNAs and catalyze their replication to dsRNAs in vitro.

Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA.

Viral infection induces the production of interleukin (IL)-1beta and IL-18 in macrophages through the activation of caspase-1, but the mechanism by which host cells sense viruses to induce caspase-1 activation is unknown. In this report, we have identified a signaling pathway leading to caspase-1 activation that is induced by double-stranded RNA (dsRNA) and viral infection that is mediated by Cryopyrin/Nalp3. Stimulation of macrophages with dsRNA, viral RNA, or its analog poly(I:C) induced the secretion of IL-1beta and IL-18 in a cryopyrin-dependent manner.

Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities.

Rotavirus NSP2 is an abundant non-structural 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.

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system.

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs.

Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein.

Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth.

Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms.

Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins.

Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae.

Rotaviruses, members of family Reoviridae, are a major cause of acute gastroenteritis of infants and young children. The rotavirus genome consists of 11 segments of double-stranded (ds)RNA and the virion is an icosahedron composed of multiple layers of protein. The virion core is formed by a layer of VP2 and contains multiple copies of the RNA-dependent RNA polymerase VP1 and the mRNA-capping enzyme VP3.

Role of the histidine triad-like motif in nucleotide hydrolysis by the rotavirus RNA-packaging protein NSP2.

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg(2+)-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2.

Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis.

The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system.

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.

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.