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.     

A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly

Rotavirus (RV) replication occurs in cytoplasmic inclusions called viroplasms whose formation requires the interactions of RV proteins NSP2 and NSP5; however, the specific role(s) of NSP2 in viroplasm assembly remains largely unknown. To study viroplasm formation in the context of infection, we characterized two new monoclonal antibodies (MAbs) specific for NSP2. These MAbs show high-affinity binding to NSP2 and differentially recognize distinct pools of NSP2 in RV-infected cells; a previously unrecognized cytoplasmically dispersed NSP2 (dNSP2) is detected by an N-terminal binding MAb, and previously known viroplasmic NSP2 (vNSP2) is detected by a C-terminal binding MAb. Kinetic experiments in RV-infected cells demonstrate that dNSP2 is associated with NSP5 in nascent viroplasms that lack vNSP2. As viroplasms mature, dNSP2 remains in viroplasms, and the amount of diffuse cytoplasmic dNSP2 increases. vNSP2 is detected in increasing amounts later in infection in the maturing viroplasm, suggesting a conversion of dNSP2 into vNSP2. Immunoprecipitation experiments and reciprocal Western blot analysis confirm that there are two different forms of NSP2 that assemble in complexes with NSP5, VP1, VP2, and tubulin. dNSP2 associates with hypophosphorylated NSP5 and acetylated tubulin, which is correlated with stabilized microtubules, while vNSP2 associates with hyperphosphorylated NSP5. Mass spectroscopy analysis of NSP2 complexes immunoprecipitated from RV-infected cell lysates show both forms of NSP2 are phosphorylated, with a greater proportion of vNSP2 being phosphorylated compared to dNSP2. Together, these data suggest that dNSP2 interacts with viral proteins, including hypophosphorylated NSP5, to initiate viroplasm formation, while viroplasm maturation includes phosphorylation of NSP5 and vNSP2.     

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.     

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. 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.     

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.     

Structural organisation of the rotavirus nonstructural protein NSP5

Rotavirus is one of the leading agents of gastroenteritis worldwide. During infection, viral factories (viroplasms) are formed. The rotavirus nonstructural proteins NSP5 and NSP2 are the major building blocks of viroplasms; however, NSP5 function and organisation remain elusive. In this report, we present a structural characterisation of NSP5. Multi-angle laser light scattering, sedimentation velocity and equilibrium sedimentation experiments demonstrate that recombinant full-length NSP5 forms a decamer in solution. Far-Western, pull-down and multi-angle laser light scattering experiments show that NSP5 has two oligomerisation regions. The first region, residues 103-146, is involved in NSP5 dimerisation, whereas the second region, residues 189-198, is responsible for NSP5 decamerisation. Circular dichroism analyses of full-length and truncated forms of NSP5 reveal that the decamerisation region is helical, whereas the dimerisation region involves β-sheets. From these circular dichroism experiments, we also show that the NSP5 protomers contain two α-helices, a disordered N-terminal half and a C-terminal half that is primarily composed of β-sheet folds. This extensive structural characterisation of NSP5 led us to propose a model for its quaternary organisation. Finally, co-expression of NSP5 fragments and NSP2 in uninfected cells shows that the NSP5 decamerisation region is required for viroplasm-like structure formation. However, in vitro, the NSP5 decamerisation region partially inhibits the NSP2-NSP5 interaction. Our NSP5 model suggests that steric hindrance prevents NSP2 from binding to all NSP5 protomers. Some protomers may thus be free to interact with other NSP5 binding partners, such as viral RNAs and the viral polymerase VP1, to perform functions other than viroplasm organisation.     

Cryoelectron microscopy structures of rotavirus NSP2-NSP5 and NSP2-RNA complexes: implications for genome replication

The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-A central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP566-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP566-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP566-188, the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.     

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms,and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.Rotaviruses are a major cause of acute gastroenteritis in infants and young children, producing a high burden of disease worldwide and over 600,000 deaths per annum, mainly in developing countries (43). Recently, two live attenuated rotavirus vaccines (49, 58) have been licensed in various countries, and their widespread use in universal mass vaccination programs is being implemented (55).Rotaviruses form a genus of the Reoviridae family. They contain a genome of 11 segments of double-stranded RNA (dsRNA) encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). After entry into the host cell the outer layer of the triple-layered particles (TLPs; infectious virions) is removed in endocytic vesicles, and the resulting double-layered particles (DLPs) actively transcribe mRNAs from the 11 RNA segments and release them into the cytoplasm. The mRNAs are translated into proteins but also act as templates for dsRNA synthesis (RNA replication). The early stages of viral morphogenesis and viral RNA replication occur in cytoplasmic inclusion bodies termed “viroplasms.” Partially assembled DLPs are released from viroplasms and receive their outer layer in the rough endoplasmic reticulum (RER), forming TLPs (for details, see Estes and Kapikian [20]).The rotavirus nonstructural proteins NSP2 and NSP5 are major components of viroplasms (20, 47). These two proteins alone are sufficient to induce the formation of viroplasm-like structures (VLS) (21). Blocking of either NSP2 or NSP5 in rotavirus-infected cells significantly reduces viroplasm formation and the production of infectious viral progeny (11, 54, 57). Until now, host cell proteins involved in viroplasm formation have not been identified.Morphological similarities between viroplasms and lipid droplets (LDs) prompted us to investigate their relationship. Both structures have phosphoproteins (NSP5 and perilipin A, respectively) inserted on their surface in ringlike shapes (16, 34). LDs are intracellular organelles involved in lipid and carbohydrate metabolism. They consist of a neutral lipid core surrounded by a phospholipid monolayer containing LD-associated proteins; those include proteins of the PAT family consisting of perilipin, adipophilin (adipose differentiation-related protein [ADRP]), and TIP-47 (9, 37). Lipolysis from LDs is regulated by hormones such as catecholamines, which trigger the phosphorylation of hormone-sensitive lipase (HSL) and perilipin A and induce LD fragmentation. Incubating adipocytes with the β-adrenergic agonist isoproterenol and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) activates this pathway (27, 34). LD formation can also be blocked by triacsin C, a specific inhibitor of long chain acyl coenzyme A synthetases (30, 39).We demonstrate here that rotavirus viroplasms colocalize with the LD-associated proteins perilipin A and ADRP and also with the lipids of LDs. These interactions appear to be required for the formation of functional viroplasms and the production of infectious viral progeny, since compounds dispersing LDs or blocking LD formation significantly decrease the number and size of viroplasms and the amount of infectious progeny. Taken together, these findings strongly suggest a critical role of LDs in rotavirus replication.     

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.     

In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms.

NSP5 (NS26), the product of rotavirus gene 11, is a phosphoprotein whose role in the virus replication cycle is unknown. To gain further insight into its function, we obtained monoclonal antibodies against the baculovirus-expressed protein. By immunoprecipitation and immunoblotting experiments, we showed that (i) NSP5 appears in many different phosphorylated forms in rotavirus-infected cells; (ii) immunoprecipitated NSP5 from rotavirus-infected cells can be phosphorylated in vitro by incubation with ATP; (iii) NSP5, produced either by transient transfection of rotavirus gene 11 or by infection by gene 11 recombinant vaccinia virus or baculovirus, can be phosphorylated in vivo and in vitro; (iv) NSP5 expressed in Escherichia coli is phosphorylated in vitro, and thus NSP5 is a potential protein kinase; and (v) NSP5 forms dimers and interacts with NSP2. The intracellular localization of NSP5 in the course of rotavirus infection and after transient expression in COS7 cells has also been investigated. In rotavirus-infected cells, NSP5 is localized in viroplasms, but it is widespread throughout the cytoplasm of transfected COS7 cells. NSP5 produced by transfected COS7 cells did not acquire the multiphosphorylated forms observed in rotavirus-infected COS7 cells. Thus, there is a tight correlation between the localization of NSP5 in the viroplasms and its protein kinase activity in vivo or in vitro. Our results suggest that cellular or viral cofactors are indispensable to fully phosphorylate NSP5 and to reach its intracellular localization.     

The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2

Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.     

Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.     

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.     

The formation of viroplasm-like structures by the rotavirus NSP5 protein is calcium regulated and directed by a C-terminal helical domain

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic alpha-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 alpha-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted alpha-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.     

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.     

Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation

Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures.     

Thiazolides,a New Class of Antiviral Agents Effective against Rotavirus Infection,Target Viral Morphogenesis,Inhibiting Viroplasm Formation

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC₅₀s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC₅₀s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against 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. 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.