Conserved rotavirus NSP5 and VP2 domains interact and affect viroplasm

One step of the life cycle common among all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of non-structural protein 5 (NSP5) with either the RV core-shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, or I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as dominant-negative by disrupting the formation of either VLSs or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV A to H species. The detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication. Importance Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients but also for adults traveling to high-risk regions and even for livestock industry. Aside from structural and physiological divergences among RV species studied until now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV proteins NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral

Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments

Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication

ABSTRACT Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication

Identification of a small molecule that compromises the structural integrity of viroplasms and rotavirus double-layered particles

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells

Dissection of mammalian orthoreovirus µ2 reveals a self-associative domain required for binding to microtubules but not to factory matrix protein µNS

Mammalian orthoreovirus protein μ2 is a component of the viral core particle. Its activities include RNA binding and hydrolysis of the γ-phosphate from NTPs and RNA 5´-termini, suggesting roles as a cofactor for the viral RNA-dependent RNA polymerase, λ3, first enzyme in 5´-capping of viral plus-strand RNAs, and/or prohibitory of RNA-5´-triphosphate-activated antiviral signaling. Within infected cells, μ2 also contributes to viral factories, cytoplasmic structures in which genome replication and particle assembly occur. By associating with both microtubules (MTs) and viral factory matrix protein μNS, μ2 can anchor the factories to MTs, the full effects of which remain unknown. In this study, a protease-hypersensitive region allowed μ2 to be dissected into two large fragments corresponding to residues 1–282 and 283–736. Fusions with enhanced green fluorescent protein revealed that these amino- and carboxyl-terminal regions of μ2 associate in cells with either MTs or μNS, respectively. More exhaustive deletion analysis defined μ2 residues 1–325 as the minimal contiguous region that associates with MTs in the absence of the self-associating tag. A region involved in μ2 self-association was mapped to residues 283–325, and self-association involving this region was essential for MT-association as well. Likewise, we mapped that μNS-binding site in μ2 relates to residues 290–453 which is independent of μ2 self-association. These findings suggest that μ2 monomers or oligomers can bind to MTs and μNS, but that self-association involving μ2 residues 283–325 is specifically relevant for MT-association during viral factories formation

Studies on rotavirus NSP5 phosphorylation and its interaction with NSP2

Rotavirus NSP5 is a non-structural protein that localises in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyper-phosphorylation , generating species with reduced PAGE mobility. This process has been suggested to be due in part to autophosphorylation. Here, we show that it rather works as an autoregulator of its own phosphorylation though the activation of cellular kinases. In this thesis, it is described the development of an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA 104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments, we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione-S-transferase-NSP5 mutants were produced in E. coli and used to determine phosphoacceptor sites. These were mapped to four serines (153, 155, 163 and 165) within an acidic region with homology to casein kinase 2 (CK2) phosphorylation sites. CK2 was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localisation in the context of the virus infection. A second hyper-phosphorylation assay was also developed. This is an in vivo assay in which , two constructs are co-transfected . One of them tagged with 11 aa served as substrate while the other was used to map the domains required to induce activation of the cellular kinase. We learn that the two activities can be separated , demonstrating that the hyper-phosphorylation is a process in trans, with one molecule activating and the other being phosphorylated. We described a motif a (from amino acids 63 to 67) in region 2 with the amino acidic sequence SDSAS. This motif, and in particular phosphorylated serine 67, is responsible to trigger the hyper-phosphorylation process. In fact, mutation of serine 67 to aspartic acid in the full-length NSP5 allowed hyper-phosphorylation of NSP5 in the absence of NSP2, suggesting that NSP2 could produce a conformational change in NSP5 to expose motif a (serine 67), thus allowing phosphorylation of serine 67. On the other hand, NSP5 substrate characteristics were mapped in region 4 (amino acids 131 to 179). The serines 153, 155, 163 and 165, that are CK2-like phosphorylation sites , are in part responsible for the hyperphosphorylation. Two other serines, 137 and 142, that are a putative sites for PKC phosphorylation are good candidates to be also substrates in NSP5 hyperphosphorylation. Moreover, the c-terminal tail (T) of NSP5 of 18 aa was found to be also necessary for activation of the cellular kinase. Although its role is not yet clear, it is possible that a dimersation in trans with another NSP5 molecule can explain the results. Alternatively, a direct interaction with a cellular kinase may be required to permit its activation Viroplasms are discrete structures formed in the cytoplasm of cells sustaining rotavirus replication that constitute the machinery of replication of the virus. In this thesis, it has been investigated the relative localisation of NSP5 and NSP2 within viroplasms as well as the dynamics of viroplasm formation in cells infected with rotavirus that also express NSP5 or NSP5 fused to EGFP. The results showed NSP2 localising more internally with respect to NSP5. The number of viroplasms was shown to first increase and then to decrease in post-infection time, while the area of each one increased , suggesting a fusion between them. The interaction between NSP2 and NSP5 was investigated using two different assays, namely a two-hybrid system and an in vivo binding assay. Both methods gave essentially the same results, indicating that in NSP5 the N-terminal region (33 aa) as well as C-terminal part (amino acids 131 to 198) are required for bind ing to NSP2. These two regions were able to confer to EGFP ability to local ise in viroplasm and to form VLS with NSP5

Mammalian orthoreovirus core protein μ2 reorganizes host microtubule-organizing center components

Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome segments, and packaging of virus progeny into newly synthesized virus cores take place. In infected cells, the MRV μ2 protein forms punctae in the enlarged region of the filamentous VFs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (MT)-extension, features common to MT-organizing centers (MTOCs). Using a previously established reconstituted VF model, we addressed the functions of MT-components and MTOCs concerning their roles in the formation of filamentous VFs. Indeed, the MTOC markers γ-tubulin and centrin were redistributed within the VF-like structures (VFLS) in a μ2-dependent manner. Moreover, the MT-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-μ2 and μNS. Thus, μ2, by interaction with γ-tubulin, can modulate MTOCs localization and function according to viral needs

The Role of the Host Cytoskeleton in the Formation and Dynamics of Rotavirus Viroplasms

Rotavirus (RV) replicates within viroplasms, membraneless electron-dense globular cytosolic inclusions with liquid–liquid phase properties. In these structures occur the virus transcription, replication, and packaging of the virus genome in newly assembled double-layered particles. The viroplasms are composed of virus proteins (NSP2, NSP5, NSP4, VP1, VP2, VP3, and VP6), single- and double-stranded virus RNAs, and host components such as microtubules, perilipin-1, and chaperonins. The formation, coalescence, maintenance, and perinuclear localization of viroplasms rely on their association with the cytoskeleton. A stabilized microtubule network involving microtubules and kinesin Eg5 and dynein molecular motors is associated with NSP5, NSP2, and VP2, facilitating dynamic processes such as viroplasm coalescence and perinuclear localization. Key post-translation modifications, particularly phosphorylation events of RV proteins NSP5 and NSP2, play pivotal roles in orchestrating these interactions. Actin filaments also contribute, triggering the formation of the viroplasms through the association of soluble cytosolic VP4 with actin and the molecular motor myosin. This review explores the evolving understanding of RV replication, emphasizing the host requirements essential for viroplasm formation and highlighting their dynamic interplay within the host cell

The dynamics of both filamentous and globular mammalian reovirus viral factories rely on the microtubule network

Mammalian reovirus viral factories (VFs) form filamentous or globular structures depending on the viral strain. In this study, we attempt to characterize the dynamics of both filamentous and globular VFs. Here, we present evidence demonstrating that globular VFs are dynamic entities coalescing between them, thereby gaining in size and concomitantly decreasing in numbers during the course of the infection. Additionally, both kinds of VFs condense into a perinuclear position. Our results show that globular VFs rely on an intact MT-network for dynamic motion, structural assembly, and maintenance and for perinuclear condensation. Interestingly, dynein localizes in both kinds of VFs, having a role at least in large globular VFs formation. To study filamentous VF dynamics, we used different transfection ratios of µNS with filamentous µ2. We found a MT-network dependency for VF-like structures perinuclear condensation. Also, µNS promotes VFLSs perinuclear positioning as well as an increase in acetylated tubulin levels