Viral infection induces the production of interleukin (IL)-1␤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 doublestranded 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-1␤ and IL-18 in a cryopyrin-dependent manner. Consistently, caspase-1 activation triggered by poly(I:C), dsRNA, and viral RNA was abrogated in macrophages lacking cryopyrin or the adaptor ASC (apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain) but proceeded normally in macrophages deficient in Toll-like receptor 3 or 7. We have also shown that infection with Sendai and influenza viruses activates the cryopyrin inflammasome. Finally, cryopyrin was required for IL-1␤ production in response to poly(I:C) in vivo. These results identify a mechanism mediated by cryopyrin and ASC that links dsRNA and viral infection to caspase-1 activation resulting in IL-1␤ and IL-18 production.

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. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.Rotaviruses, members of the Reoviridae, are an important cause of acute gastroenteritis in infants and young children (15). The virion is an icosahedron composed of three concentric layers of protein with a genome of 11 segments of doublestranded RNA (dsRNA) (30). The outer layer of the infectious triple-layered particle (TLP) is made up of the glycoprotein, VP7, and the spike protein, VP4. The intermediate layer is formed by VP6 trimers, and the inner layer is formed by the core lattice protein, VP2, arranged with Tϭ1 icosahedral symmetry. Positioned at the vertices of the VP2 lattice are individual copies of the RNA-dependent RNA polymerase (RdRp) VP1, and the mRNA-capping enzyme VP3. Together, VP1, VP2, VP3, and the dsRNA genome make up the core of the virion (19).Rotavirus entry is accompanied by the loss of the VP4 and VP7 outer layer, thereby converting TLPs to double-layered particles (DLPs). The RdRp of the DLP functions as a transcriptase to synthesize the 11 viral plus-strand RNAs (18). The plus-strand RNAs are extruded from DLPs through channels at the vertices that extend through both the VP2 and VP6 protein layers. The plus-strand RNAs contain 5Ј caps but lack 3Ј poly(A) tails and are translated to gi...

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. Our sequencing results revealed that most of the individual proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral protein, many of which are sites of known protein-protein interactions. For the intermediate viral capsid protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral proteins during replication.

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.

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The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds singlestranded RNA cooperatively, with high affinity, and independent of sequence. Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear. In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy. We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP. In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed. This was reversed by binding of ADP and RNA. We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation. The secondary structure of NSP2 showed a high fraction of ␤-sheet, Rotavirus is a significant cause of disease in humans and animals. It is a member of the Reoviridae, and its genome consists of 11 segments of double-stranded RNA, which codes for six structural and six nonstructural proteins. The structural proteins include VP2, VP6, and VP7, which form the triple layered icosahedral virion capsid, the spike protein VP4, the RNA polymerase VP1, and the multifunctional capping enzyme VP3 (1) (for a review, see Ref.2). Many studies have addressed the properties of the structural proteins, for example their spatial configuration in the virus particle using cryo-EM (3, 4), their antigenicity, and their role in viral entry, replication, or morphogenesis. Unfortunately, much less is known about the nonstructural proteins that are expressed and left behind in the infected cells.Although it has been shown that these nonstructural proteins are not essential for replicase activity in vitro (5), they are important in several aspects of the replication cycle of the virus in vivo. Some of the more intensively studied nonstructural proteins include, for example, NSP4, which has a membranedestabilizing activity and assists in the budding of newly synthesized inner capsid particles into the lumen of the endoplasmic reticulum (6, 7), where they acquire the outer coat protein VP7. NSP3 binds to the 3Ј-end of the viral mRNA and interacts with eukaryotic translation initiation factor eIF4G to enhance efficiency of translation (8). NSP2 and NSP5 interact to form viroplasms, large inclusions in the cytoplasm where core-like replication intermediates (core RIs) 1 are assembled and RNA replication takes place (2, 9, 10). As was shown in a study of a temperature-sensitive mutant, NSP2 is required for the formation of the viroplasm and is also essential in the synthesis of double-stranded RNA in vivo (11). However, little is known about the mechanism underlying this observation and the function of NSP2 on a molecular level.NSP2 is a 35-kDa protein that forms homomultimers and interacts with the RNA polymerase VP1 (12, 17). F...

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. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ≥10 4 pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this "two-hit" reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.recombinant virus | Reoviridae | NSP2 | RNAi | temperature-sensitive virus T he greatest obstacle preventing translation of basic research to rotavirus (RV) biology is the relative inability to manipulate the 11-segmented, double-stranded RNA (dsRNA) viral genome through reverse genetics. Moreover, RV is a significant human pathogen, and an unencumbered reverse genetics system would allow the development of recombinant vaccine strains and could be used to further our understanding of RV transmission, pathogenesis, and immunity. Although it is possible to engineer RV strains containing a single recombinant gene, the previously described methods have significant limitations in terms of the genes that can be manipulated and the efficiency of recombinant virus recovery. Our objective in this study was to develop an efficient, broadly applicable method to generate recombinant RVs.Recent advances for orthoreovirus (reovirus) and bluetongue virus have demonstrated that it is possible to reconstitute other Reoviridae viruses entirely from recombinant sources (1-5). RV, conversely, has remained recalcitrant to similar techniques and is limited to helper-virus-dependent, single-gene replacement methods. The prototypic RV reverse genetics technique uses neutralizing monoclonal antibody (N-mAb) selection against a helper RV strain to isolate a single-gene recombinant virus (6). This method can be used to manipulate the genome segments (genes) that encode the outer-capsid proteins of RV: VP4 and VP7. N-mAb-based reverse genetics has yielded information about the properties of VP4 neutralization and how it acts as a determinant of spread in cell culture (7), but this approach has not be...

The rotavirus nonstructural protein NSP2 self-assembles into homomultimers, binds single-stranded RNA nonspecifically, possesses a Mg 2؉ -dependent nucleoside triphosphatase (NTPase) activity, and is a component of replication intermediates. Because these properties are characteristics of known viral helicases, we examined the possibility that this was also an activity of NSP2 by using a strand displacement assay and purified bacterially expressed protein. The results revealed that, under saturating concentrations, NSP2 disrupted both DNA-RNA and RNA-RNA duplexes; hence, the protein possesses helix-destabilizing activity. However, unlike typical helicases, NSP2 required neither a divalent cation nor a nucleotide energy source for helix destabilization. Further characterization showed that NSP2 displayed no polarity in destabilizing a partial duplex. In addition, helix destabilization by NSP2 was found to proceed cooperatively and rapidly. The presence of Mg 2؉ and other divalent cations inhibited by approximately one-half the activity of NSP2, probably due to the increased stability of the duplex substrate brought on by the cations. In contrast, under conditions where NSP2 functions as an NTPase, its helix-destabilizing activity was less sensitive to the presence of Mg 2؉ , suggesting that in the cellular environment the two activities associated with the protein, helix destabilization and NTPase, may function together. Although distinct from typical helicases, the helix-destabilizing activity of NSP2 is quite similar to that of the NS protein of reovirus and to the single-stranded DNA-binding proteins (SSBs) involved in double-stranded DNA replication. The presence of SSB-like nonstructural proteins in two members of the family Reoviridae suggests a common mechanism of unwinding viral mRNA prior to packaging and subsequent minus-strand RNA synthesis.Rotaviruses, members of the family Reoviridae, are the major cause of severe gastroenteritis in infants and young children (16). The viruses are icosahedrons made up of three concentric layers of protein and contain a genome composed of eleven segments of double-stranded RNA (dsRNA) (8). The outermost capsid layer consists of the spike protein, VP4, and the glycoprotein, VP7, and the intermediate layer is formed by VP6 trimers (34). The innermost layer consists of 60 dimers of VP2, arranged as a Tϭ1 icosahedron (21). Positioned at the vertices of the VP2 icosahedron are one copy each of the RNA-dependent RNA polymerase (RdRP), VP1, and the capping enzyme, VP3 (21). Together, VP1, VP2, VP3, and the dsRNA genome make up the core of the virion. Double-layered particles, representing cores surrounded by VP6, have an associated transcriptase activity that catalyzes the synthesis of viral mRNAs (6, 22). The mRNAs not only direct protein synthesis but also serve as templates for the synthesis of minus-strand RNA to form dsRNA (5). Minus-strand synthesis occurs soon after or as viral mRNAs are packaged into core-like replication intermediates (RIs) (9, 32). In addition to t...