Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories.

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.

The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication.

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.

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.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.

A Single Nucleoside Viral Polymerase Inhibitor Against Norovirus, Rotavirus, and Sapovirus-Induced Diarrhea.

A safe and highly efficient antiviral is needed for the prophylaxis and/or treatment of viral diarrhea. We here demonstrate the in vitro antiviral activity of four 2'-C-methyl nucleoside analogues against noro-, rota-, and sapoviruses. The most potent nucleoside analogue, 7-deaza-2'-C-methyladenosine, inhibits replication of these viruses with a 50% effective concentration < 5 µM. Mechanistically, we demonstrate that the 2'-C-methyl nucleoside analogues act by inhibiting transcription of the rotavirus genome. This provides the first evidence that a single viral-diarrhea-targeted treatment can be developed through a viral-polymerase-targeting small molecule.

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.

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.

In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system.

BACKGROUND: Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused to a 15 amino acid long Biotin Acceptor Peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin to the single lysine residue of the BAP sequence. However, this system is mainly based on the contemporaneous use of two different plasmids or on induction of expression of two proteins through an IRES-driven mechanism. RESULTS: We developed a single bigenic plasmid that contains two independent transcriptional units for the co-expression of both the protein tagged with BAP and an engineered version of the BirA enzyme. Upstream of the cDNA encoding BirA, a signal secretion leader sequence was added to allow translocation of the enzyme to the secretory pathway. Three different recombinant antibodies in the scFv format, a membrane bound and secretory truncated IgE Fc fragment and a soluble version of the human IgE high affinity receptor were shown to be efficiently biotinylated and to maintain their binding properties in immunofluorescence microscopy, flow cytometry and ELISA assays. CONCLUSION: The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific in vivo biotinylation of target molecules tagged with a short acceptor peptide. These molecules could be easily obtained from supernatants or extracts of mammalian cells and used for a wide range of biological applications.

VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9.

The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology.

Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle.

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.

DNA-immunisation with dengue virus E protein domains I/II, but not domain III, enhances Zika, West Nile and Yellow Fever virus infection.

Dengue virus (DENV), the causative agent of dengue disease, is among the most important mosquito-borne pathogens worldwide. DENV is composed of four closely related serotypes and belongs to the Flaviviridae family alongside other important arthropod-borne viral pathogens such as Zika virus (ZIKV), West Nile virus (WNV) and Yellow Fever virus (YFV). After infection, the antibody response is mostly directed to the viral E glycoprotein which is composed of three structural domains named DI, DII and DIII that share variable degrees of homology among different viruses. Recent evidence supports a close serological interaction between ZIKV and DENV. The possibility of worse clinical outcomes as a consequence of antibody-dependent enhancement of infection (ADE) due to cross-reactive antibodies with poor neutralisation activity is a matter of concern. We tested polyclonal sera from groups of female Balb/C mice vaccinated with DNA constructs expressing DI/DII, DIII or the whole sE from different DENV serotypes and compared their activity in terms of cross-reactivity, neutralisation of virus infection and ADE. Our results indicate that the polyclonal antibody responses against the whole sE protein are highly cross-reactive with strong ADE and poor neutralisation activities due to DI/DII immunodominance. Conversely, anti-DIII polyclonal antibodies are type-specific, with no ADE towards ZIKV, WNV and YFV, and strong neutralisation activity restricted only to DENV.

An Inhibitory Motif on the 5'UTR of Several Rotavirus Genome Segments Affects Protein Expression and Reverse Genetics Strategies.

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.

New tags for recombinant protein detection and O-glycosylation reporters.

Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation.

Rotavirus increases levels of lipidated LC3 supporting accumulation of infectious progeny virus without inducing autophagosome formation.

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 viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules.

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.

Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1alpha is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication.

The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in 'viroplasms', cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1alpha is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1alpha is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1alpha was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1alpha is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection.