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Discusses a model of an enzyme reaction cycle that includes the generation of a transient-spin correlated radical pair state. Alteration of the recombinant yield of the radical pair state by external magnetic fields; Response behavior of the enzyme to pulsed magnetic fields; Combinations of static and sinusoidally oscillating magnetic fields.

Rotaviruses (RVs) are classified into nine species, A-D and F-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions composed of main building block proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 in uninfected cells leads to the formation of viroplasm-like structures (VLSs). Although morphologically identical to viroplasms, VLSs do not produce viral progeny but serve as excellent tools for studying complex viroplasms. A knowledge gap exists regarding non-RVA viroplasms due to the lack of specific antibodies and suitable cell culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from RVJ, all NSP5 interacted with their cognate NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2., Importance: Rotaviruses (RVs) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLSs) by the co-expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with their cognates NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is required for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species., Competing Interests: The authors declare no conflict of interest.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schramm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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., Competing Interests: The authors declare no conflict of interest.

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.

Rotavirus (RV) replication occurs in cytoplasmic membrane-less, electron-dense inclusions termed viroplasms, composed of viral and cellular elements. These inclusions have been shown to colocalize with components of the lipid droplets (LDs), unique organelles that play an essential role in lipid metabolism. Given the robust LDs-viroplasm association, LDs have been proposed to serve as a scaffold for viroplasm assembly. Interestingly, no evidence has described the participation of lipid metabolism in other RV replication steps. Here, we report that lipid metabolism is essential to maintain the production of the infectious virus through a process independent of viroplasm biogenesis. Disruption of the lipogenesis-lipolysis balance dissociates endoplasmic reticulum membranes from viroplasms, suggesting that lipid metabolism is essential for a continuous flux of lipids to allow the association between viroplasms and ER membranes. LDs could also be relevant as lipid reservoirs for membrane synthesis required to form mature infectious rotavirus particles., (Copyright © 2022 Elsevier Inc. All rights reserved.)

One of the foremost goals in vaccine development is the design of effective, heat-stable vaccines that simplify the distribution and delivery while conferring high levels of protective immunity. Here, we describe a method for developing a live, oral vaccine that relies on the biofilm-forming properties of the spore-former bacterium Bacillus subtilis. The amyloid protein TasA is an abundant component of the extracellular matrix of the biofilms formed by B. subtilis that can be genetically fused to an antigen of interest. Spores of the recombinant strain are then prepared and applied via the oral route in an animal model. Due to the intrinsic resistance of the spores, they can bypass the stomach barrier, germinate, and subsequently colonize the gut, where they develop into biofilms, expressing the antigen of interest. We describe here the steps necessary to produce spores, immunization, and downstream analysis of the vaccine efficacy., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

One step of the life cycle common to 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 nonstructural protein 5 (NSP5) with 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, and 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 a dominant-negative element by disrupting the formation of either VLS or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV species A to H. 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, adults traveling to high-risk regions, and even for the livestock industry. Aside from structural and physiological divergences among RV species studied before 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 protein 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., (Copyright © 2020 American Society for Microbiology.)

Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, although how they assemble remains unknown. During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue. The role of this posttranslational modification in the formation of viroplasms and its impact on virus replication remain obscure. Here, we investigated the role of NSP5 during RV infection by taking advantage of a modified fully tractable reverse-genetics system. A trans -complementing cell line stably producing NSP5 was used to generate and characterize several recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that an rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication. A number of mutants with impaired NSP5 phosphorylation were generated to further interrogate the function of this posttranslational modification in the assembly of replication-competent viroplasms. We showed that the rRV mutant strains exhibited impaired viral replication and the ability to assemble round-shaped viroplasms in MA104 cells. Furthermore, we investigated the mechanism of NSP5 hyperphosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, as well as MA104-derived stable transfectant cell lines expressing either wild-type NSP5 or selected NSP5 deletion mutants. Our results indicate that NSP5 hyperphosphorylation is a crucial step for the assembly of round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 in the formation of replication-competent viral factories. Such a complex NSP5 phosphorylation cascade may serve as a paradigm for the assembly of functional viral factories in other RNA viruses. IMPORTANCE The rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The nonstructural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully tractable reverse-genetics (RG) system for rotaviruses. Here, we show a novel application of a recently developed RG system by establishing a stable trans -complementing NSP5-producing cell line required to rescue rotaviruses with mutations in NSP5. This approach allowed us to provide the first direct evidence of the pivotal role of this protein during RV replication. Furthermore, using recombinant RV mutants, we shed light on the molecular mechanism of NSP5 hyperphosphorylation during infection and its involvement in the assembly and maturation of replication-competent viroplasms., (Copyright © 2019 Papa et al.)

Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4. IMPORTANCE Rotavirus, a member of the family Reoviridae , is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4., (Copyright © 2019 American Society for Microbiology.)

Background: We previously engineered Bacillus subtilis to express an antigen of interest fused to TasA in a biofilm. B. subtilis has several properties such as sporulation, biofilm formation and probiotic ability that were used for the oral application of recombinant spores harboring Echinococcus granulosus paramyosin and tropomyosin immunogenic peptides that resulted in the elicitation of a specific humoral immune response in a dog model., Results: In order to advance our understanding of the research in oral immunization practices using recombinant B. subtilis spores, we describe here an affordable animal model. In this study, we show clear evidence indicating that a niche is required for B. subtilis recombinant spores to colonize the densely populated mice intestinal microbiota. The reduction of intestinal microbiota with an antibiotic treatment resulted in a positive elicitation of local humoral immune response in BALB/c mice after oral application of recombinant B. subtilis spores harboring TasA fused to E. granulosus (102-207) EgTrp immunogenic peptide. Our results were supported by a lasting prevalence of spores in mice feces up to 50 days after immunization and by the presence of specific secretory IgA, isolated from feces, against E. granulosus tropomyosin., Conclusions: The reduction of mouse intestinal microbiota allowed the elicitation of a local humoral immune response in mice after oral application with spores of B. subtilis harboring immunogenic peptides against E. granulosus.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During the late stages of rotavirus morphogenesis, the surface proteins VP4 and VP7 are assembled onto the previously structured double-layered virus particles to yield a triple-layered, mature infectious virus. The current model for the assembly of the outer capsid is that it occurs within the lumen of the endoplasmic reticulum. However, it has been shown that VP4 and infectious virus associate with lipid rafts, suggesting that the final assembly of the rotavirus spike protein VP4 involves a post-endoplasmic reticulum event. In this work, we found that the actin inhibitor jasplakinolide blocks the cell egress of rotavirus from nonpolarized MA104 cells at early times of infection, when there is still no evidence of cell lysis. These findings contrast with the traditional assumption that rotavirus is released from nonpolarized cells by a nonspecific mechanism when the cell integrity is lost. Inspection of the virus present in the extracellular medium by use of density flotation gradients revealed that a fraction of the released virus is associated with low-density membranous structures. Furthermore, the intracellular localization of VP4, its interaction with lipid rafts, and its targeting to the cell surface were shown to be prevented by jasplakinolide, implying a role for actin in these processes. Finally, the VP4 present at the plasma membrane was shown to be incorporated into the extracellular infectious virus, suggesting the existence of a novel pathway for the assembly of the rotavirus spike protein. IMPORTANCE Rotavirus is a major etiological agent of infantile acute severe diarrhea. It is a nonenveloped virus formed by three concentric layers of protein. The early stages of rotavirus replication, including cell attachment and entry, synthesis and translation of viral mRNAs, replication of the genomic double-stranded RNA (dsRNA), and the assembly of double-layered viral particles, have been studied widely. However, the mechanisms involved in the later stages of infection, i.e., viral particle maturation and cell exit, are less well characterized. It has been assumed historically that rotavirus exits nonpolarized cells following cell lysis. In this work, we show that the virus exits cells by a nonlytic, actin-dependent mechanism, and most importantly, we describe that VP4, the spike protein of the virus, is present on the cell surface and is incorporated into mature, infectious virus, indicating a novel pathway for the assembly of this protein., (Copyright © 2018 American Society for Microbiology.)

Bacillus subtilis is known as an endospore- and biofilm-forming bacterium with probiotic properties. We have recently developed a method for displaying heterologous proteins on the surface of B. subtilis biofilms by introducing the coding sequences of the protein of interest into the bacterial genome to generate a fusion protein linked to the C terminus of the biofilm matrix protein TasA. Although B. subtilis is a regular component of the gut microflora, we constructed a series of recombinant B. subtilis strains that were tested for their ability to be used to immunize dogs following oral application of the spores. Specifically, we tested recombinant spores of B. subtilis carrying either the fluorescent protein mCherry or else selected antigenic peptides (tropomyosin and paramyosin) from Echinococcus granulosus , a zoonotic intestinal tapeworm of dogs and other carnivores. The application of the recombinant B. subtilis spores led to the colonization of the gut with recombinant B. subtilis but did not cause any adverse effect on the health of the animals. As measured by enzyme-linked immunosorbent assay and immunoblotting, the dogs were able to develop a humoral immune response against mCherry as well as against E. granulosus antigenic peptides. Interestingly, the sera of dogs obtained after immunization with recombinant spores of E. granulosus peptides were able to recognize E. granulosus protoscoleces, which represent the infective form of the head of the tapeworms. These results represent an essential step toward the establishment of B. subtilis as an enteric vaccine agent., (Copyright © 2018 American Society for Microbiology.)

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. IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection., (Copyright © 2018 American Society for Microbiology.)

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.

In infected cells rotavirus (RV) replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT) network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2) involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.

Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5'UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5'UTRs of eight genome segments (gs3, gs5-6, gs7-11) of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3'UTR sequences. Analysis of several mutants of the 21-nucleotide long 5'UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5' terminal 6-nucleotide long pyrimidine-rich tract 5'-GGY(U/A)UY-3'. The 5' terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5'UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies., Competing Interests: The authors have declared that no competing interests exist.

Background: Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins., Results: We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties., Conclusions: In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Replication of many RNA viruses benefits from subversion of the autophagic pathway through many different mechanisms. Rotavirus, the main etiologic agent of pediatric gastroenteritis worldwide, has been recently described to induce accumulation of autophagosomes as a mean for targeting viral proteins to the sites of viral replication. Here we show that the viral-induced increase of the lipidated form of LC3 does not correlate with an augmented formation of autophagosomes, as detected by immunofluorescence and electron microscopy. The LC3-II accumulation was found to be dependent on active rotavirus replication through the use of antigenically intact inactivated viral particles and of siRNAs targeting viral genes that are essential for viral replication. Silencing expression of LC3 or of Atg7, a protein involved in LC3 lipidation, resulted in a significant impairment of viral titers, indicating that these elements of the autophagic pathway are required at late stages of the viral cycle.

Rotavirus viroplasms are cytosolic, electron-dense inclusions corresponding to the viral machinery of replication responsible for viral template transcription, dsRNA genome segments replication and assembly of new viral cores. We have previously observed that, over time, those viroplasms increase in size and decrease in number. Therefore, we hypothesized that this process was dependent on the cellular microtubular network and its associated dynamic components. Here, we present evidence demonstrating that viroplasms are dynamic structures, which, in the course of an ongoing infection, move towards the perinuclear region of the cell, where they fuse among each other, thereby gaining considerably in size and, simultaneously, explaining the decrease in numbers. On the viral side, this process seems to depend on VP2 for movement and on NSP2 for fusion. On the cellular side, both the temporal transition and the maintenance of the viroplasms are dependent on the microtubular network, its stabilization by acetylation, and, surprisingly, on a kinesin motor of the kinesin-5 family, Eg5. Thus, we provide for the first time deeper insights into the dynamics of rotavirus replication, which can explain the behavior of viroplasms in the infected cell.

Expression of a high-risk human papillomavirus (HPV) E7 oncoprotein is sufficient to induce aberrant centrosome duplication in primary human cells. The resulting centrosome-associated mitotic abnormalities have been linked to the development of aneuploidy. HPV type 16 (HPV16) E7 induces supernumerary centrosomes through a mechanism that is at least in part independent of the inactivation of the retinoblastoma tumor suppressor pRb and is dependent on cyclin-dependent kinase 2 activity. Here, we show that HPV16 E7 can concentrate around mitotic spindle poles and that a small pool of HPV16 E7 is associated with centrosome fractions isolated by sucrose density gradient centrifugation. The targeting of HPV16 E7 to the centrosome, however, was not sufficient for centrosome overduplication. Nonetheless, we found that HPV16 E7 can associate with the centrosomal regulator gamma-tubulin and that the recruitment of gamma-tubulin to the centrosome is altered in HPV16 E7-expressing cells. Since the association of HPV16 E7 with gamma-tubulin is independent of pRb, p107, and p130, our results suggest that the association with gamma-tubulin contributes to the pRb/p107/p130-independent ability of HPV16 E7 to subvert centrosome homeostasis.

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein muNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase lambda3, that was recruited to the structures through an association with muNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of muNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the muNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent muNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.

Rotavirus genomes contain 11 double-stranded (ds) RNA segments. Genome segment 11 encodes the non-structural protein NSP5 and, in some strains, also NSP6. NSP5 is produced soon after viral infection and localizes in cytoplasmic viroplasms, where virus replication takes place. RNA interference by small interfering (si) RNAs targeted to genome segment 11 mRNA of two different strains blocked production of NSP5 in a strain-specific manner, with a strong effect on the overall replicative cycle: inhibition of viroplasm formation, decreased production of other structural and non-structural proteins, synthesis of viral genomic dsRNA and production of infectious particles. These effects were shown not to be due to inhibition of NSP6. The results obtained strengthen the importance of secondary transcription/translation in rotavirus replication and demonstrate that NSP5 is essential for the assembly of viroplasms and virus replication.

Rotavirus NSP5 is a nonstructural protein that localizes in viroplasms of virus-infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced PAGE mobility. Here we show that NSP5 operates as an autoregulator of its own phosphorylation as a consequence of two distinct activities of the protein: substrate and activator. We developed an in vivo hyperphosphorylation assay in which two NSP5 mutant constructs are cotransfected. One of them, fused to an 11-aa tag, served as substrate whereas the other was used to map NSP5 domains required for activation. The activation and substrate activity could be uncoupled, demonstrating a hyperphosphorylation process in trans between the activator and substratum. This process involved dimerization of the two components through the 18-aa C-terminal tail. Phosphorylation of Ser-67 within the SDSAS motif (amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation function. We present evidence of casein kinase 1alpha being the protein kinase responsible for this key step as well as for the consecutive ones leading to NSP5 hyperphosphorylation.

Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131-198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.

Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 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 Escherichia coli and used to determine phosphoacceptor sites. These were mapped to four serines (Ser(153), Ser(155), Ser(163), and Ser(165)) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII 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 localization in the context of virus infection.