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.     

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. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.     

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. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the gamma-beta 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 gamma-beta phosphoanhydride bond. Our results show that NSP2 hydrolyzes the gammaP from RNAs and NTPs through Mg(2+)-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 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 RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5'-terminal gammaP on the (-) strands of the double-stranded RNA 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 gammaP hydrolysis.     

Rotavirus nonstructural protein NSP2 self-assembles into octamers that undergo ligand-induced conformational changes

The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds single-stranded 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 beta-sheet, with small changes induced by magnesium that were reversed in the presence of RNA. That NSP2 can exist in different conformations lends support to the previously proposed hypothesis (Taraporewala, Z., Chen, D., and Patton, J. T. (1999) J. Virol. 73, 9934-9943) of its function as a molecular motor involved in the packaging of viral mRNA.     

Sequestration of Free Tubulin Molecules by the Viral Protein NSP2 Induces Microtubule Depolymerization during Rotavirus Infection

Microtubules, components of the cell cytoskeleton, play a central role in cellular trafficking. Here we show that rotavirus infection leads to a remodeling of the microtubule network together with the formation of tubulin granules. While most microtubules surrounding the nucleus depolymerize, others appear packed at the cell periphery. In microtubule depolymerization areas, tubulin granules are observed; they colocalize with viroplasms, viral compartments formed by interactions between rotavirus proteins NSP2 and NSP5. With purified proteins, we show that tubulin directly interacts in vitro with NSP2 but not with NSP5. The binding of NSP2 to tubulin is independent of its phosphatase activity. The comparison of three-dimensional (3-D) reconstructions of NSP2 octamers alone or associated with tubulin reveals electron densities in the positively charged grooves of NSP2 that we attribute to tubulin. Site-directed mutagenesis of NSP2 and competition assays between RNA and tubulin for NSP2 binding confirm that tubulin binds to these charged grooves of NSP2. Although the tubulin position within NSP2 grooves cannot be precisely determined, the tubulin C-terminal H12 α-helix could be involved in the interaction. NSP2 overexpression and rotavirus infection produce similar effects on the microtubule network. NSP2 depolymerizes microtubules and leads to tubulin granule formation. Our results demonstrate that tubulin is a viroplasm component and reveal an original mechanism. Tubulin sequestration by NSP2 induces microtubule depolymerization. This depolymerization probably reroutes the cell machinery by inhibiting trafficking and functions potentially involved in defenses to viral infections.Microtubules (MTs) are components of the cell cytoskeleton and play a major role in cellular trafficking. Molecular motors (dynein and kinesins) use MTs as tracks to address organelles to precise loci. Viruses are irreplaceable tools to study cellular processes; for example, many of them hijack cellular transport to reach the perinuclear region (for reviews, see references 27, 35, 39, and 40). Some viruses also modify the cell compartmentation and create viral inclusions where viral replication and virion assembly are performed (for a review, see reference 30). Both aspects are sometimes related; electron and fluorescence microscopy observations of reovirus-infected cells have shown that viral inclusions form an electron-dense coat surrounding the MTs (15, 32, 41). In the case of rotavirus, another member of the Reoviridae family, interactions between viral proteins and MTs remain unclear; some studies report an interaction between MTs and either NSP4 or VP4, whereas others did not detect these interactions (4, 19, 29, 51). Rotavirus is the leading agent of gastroenteritis in young children worldwide (31); studying its interactions with its host cell is thus of particular interest to identify new potential therapeutical targets.The rotavirus genome is composed of 11 double-stranded RNAs (dsRNA) surrounded by a triple-layer capsid. During rotavirus infection, punctuate cytoplasmic structures, named “viroplasms,” are formed; they are the sites of viral genome replication and virion assembly. These structures are made of several viral proteins and of viral mRNAs that serve as templates for genome replication. Two viral nonstructural proteins, NSP2 and NSP5, are crucial for viroplasm formation (10, 24, 38). Their coexpression in uninfected cells leads to the formation of punctuate cytoplasmic structures termed viroplasm-like structures (VLS) (18). NSP2 forms a doughnut-shaped octamer by tail-to-tail interaction of two tetramers; four positively charged grooves crossing the two tetramers have been identified (21). Structural and biochemical studies have revealed a histidine-triad (HIT)-like motif responsible for the nucleoside triphosphatase (NTPase), RNA triphosphatase (RTPase), and nucleoside diphosphate (NDP) kinase-like activities of NSP2 (21, 23, 42, 46). These catalytic activities are required for dsRNA synthesis but not for viroplasm formation (11, 43). NSP2 binds single-stranded RNA nonspecifically, has helix destabilizing activity (44), and undertakes conformational changes upon nucleotide binding (37). NSP2 might thus function as a molecular motor involved in genome replication and packaging. NSP5 is a dimeric O-linked glyco- and phosphoprotein, which exists as variously phosphorylated isoforms (1, 36, 48). A cryoelectron microscopy study pointed out that RNA and NSP5 compete for binding to the grooves of the NSP2 octamer (22). The function of NSP5 in rotavirus replication and the role of its phosphorylation remain unknown. No cellular partner of these two nonstructural proteins was known, until a possible association of both proteins with tubulin was reported (9).In the present report, we studied the interaction of rotavirus with tubulin and MTs. We focused on the cellular effects and the structural characterization of the interaction between tubulin and NSP2. Our results highlight that infection by the rotavirus RF strain disorganizes and depolymerizes the MT network of MA104 cells and that viroplasms colocalize with tubulin granules. Electron microscopy and biochemical experiments demonstrate that tubulin directly binds to the positively charged grooves of NSP2. Moreover, NSP2 overexpression induces MT depolymerization and tubulin granule formation. We thus propose that NSP2 sequesters tubulin in viroplasms during rotavirus infection. This sequestration induces the MT depolymerization observed during rotavirus infection and most probably modifies cellular trafficking.     

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.     

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. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-A-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (H?H?H?? where ? is a hydrophobic residue), our analyses demonstrate that histidines (His(221) and His(225)) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.     

Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity

The nonstructural protein NSP2 is a component of rotavirus replication intermediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better understand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purified form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 +/- 2 subunits of recombinant NSP2 (rNSP2). As shown by gel mobility shift assays, the rNSP2 multimers bound to RNA in discrete cooperative steps to form higher-order RNA-protein complexes. The RNA-binding activity of the rNSP2 multimers was determined to be nonspecific and to have a strong preference for single-stranded RNA over double-stranded RNA, for which it displayed little affinity. Enzymatic analysis revealed that rNSP2 possessed an associated nucleoside triphosphatase (NTPase) activity in vitro, which in the presence of Mg(2+) catalyzed the hydrolysis of each of the four NTPs to NDPs with equal efficiency. Evidence indicating that the hydrolysis of NTP resulted in the covalent linkage of the gamma-phosphate to rNSP2 was obtained. Additional experiments showed that NSP2 expressed transiently in MA014 cells is phosphorylated. We propose that NSP2 functions as a molecular motor, catalyzing the packaging of viral mRNA into core-like replication intermediates through the energy derived from its NTPase activity.     

Rotavirus nonstructural protein NSP5 interacts with major core protein VP2

Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.     

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.     

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.     

Interferon regulatory factor 3 is a cellular partner of rotavirus NSP1

The rotavirus nonstructural protein NSP1 is the least conserved protein in the rotavirus genome, and its function in the replication cycle is not known. We employed NSP1 as bait in the yeast two-hybrid interaction trap to identify candidate cellular partners of NSP1 that may provide clues to its function. Interferon regulatory factor 3 (IRF-3) was identified as an NSP1 interactor. NSP1 synthesized in rotavirus-infected cells bound IRF-3 in a glutathione S-transferase pull-down assay, indicating that the interaction was not unique to the two-hybrid system. NSP1 of murine rotavirus strain EW also interacted with IRF-3. NSP1 deletion and point mutants were constructed to map domains important in the interaction between NSP1 and IRF-3. The data suggest that a binding domain resides in the C terminus of NSP1 and that the N-terminal conserved zinc finger is important but not sufficient to mediate binding to IRF-3. We predict that a role for NSP1 in rotavirus-infected cells is to inhibit activation of IRF-3 and diminish the cellular interferon response.     

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3' termini ended with 5'-UGAACC-3' instead of the 3' consensus sequence 5'-UGACC-3', which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3' consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3' consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3' end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3' consensus sequence.     

Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.     

Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.     

Four nucleotides are the minimal requirement for RNA recognition by rotavirus non-structural protein NSP3.

The interaction of the group A rotavirus non-structural protein NSP3 (NSP3A) with RNA has been studied in vitro. Using semi-purified NSP3A protein expressed by a recombinant baculovirus and in vitro synthesized RNA, we determined by UV cross-linking and gel retardation assays that NSP3A binds, in a sequence-specific manner, the consensus sequence (AUGUGACC) present on the 3' ends of all group A rotavirus mRNAs. Using short oligoribonucleotides, we established that the minimal RNA sequence required for binding of NSP3A is GACC. Modifications of the UGACC oligonucleotide sequence impaired binding of the protein to the RNA. Furthermore, the recombinant NSP3 protein from rotavirus group C showed specificity for the 3' end consensus sequence (AUGUGGCU) of only group C mRNAs. Sequence analysis of the NSP3 proteins did not reveal significant homologies with other RNA binding proteins, thus the NSP3 proteins of rotaviruses are the prototypes of a new kind of sequence-specific RNA binding protein.     

Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.     

Identification and characterization of the helix-destabilizing activity of rotavirus nonstructural protein NSP2

The rotavirus nonstructural protein NSP2 self-assembles into homomultimers, binds single-stranded RNA nonspecifically, possesses a Mg2+-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 Mg2+ 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 Mg2+, 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 sigmaNS 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.     

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. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.