scholarly journals Measles Virus Forms Inclusion Bodies with Properties of Liquid Organelles

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Yuqin Zhou ◽  
Justin M. Su ◽  
Charles E. Samuel ◽  
Dzwokai Ma
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Virus Replication ◽  
Inclusion Bodies ◽  
Measles Virus ◽  
Rna Replication ◽  
Infected Cells ◽  
Viral Inclusion ◽  
P Proteins ◽  
Liquid Liquid Phase Separation

ABSTRACT Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells. IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally ∼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.

scholarly journals Respiratory syncytial virus sequesters NF-κB subunit p65 to cytoplasmic inclusion bodies to inhibit innate immune signalling

2020 ◽  
Author(s):  
Fatoumatta Jobe ◽  
Jennifer Simpson ◽  
Philippa Hawes ◽  
Efrain Guzman ◽  
Dalan Bailey
Keyword(s):  
Electron Microscopy ◽  
Phase Separation ◽  
Respiratory Syncytial Virus ◽  
Liquid Phase ◽  
Inclusion Bodies ◽  
Rna Replication ◽  
Innate Immune ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Liquid Liquid Phase Separation

AbstractViruses routinely employ strategies to prevent the activation of innate immune signalling in infected cells. RSV is no exception, encoding two accessory proteins (NS1 and NS2) which are well established to block Interferon signalling. However, RSV-encoded mechanisms for inhibiting NF-κB signalling are less well characterised. In this study we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins, and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV infected cells we demonstrated that the P65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, puncta which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured P65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following TNF-α stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalise RSV N protein and P65 within bRSV IBs; granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterisation of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signalling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate – a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells.Impact summaryMany viruses replicate almost entirely in the cytoplasm of infected cells, without too many direct interactions with the nucleus. Examples include respiratory syncytial virus (RSV), measles, Ebola and Nipah; however, how these pathogens are able to compartmentalise their life cycle to provide favourable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterised. In this paper we show that bovine RSV (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. These organelles are unusually membrane-less; likely forming by a process called liquid-liquid phase separation which involves macro-molecular interactions between the viral proteins N and P. We also showed that these organelles, otherwise known as inclusion bodies (IBs), are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signalling processes that tell the nucleus the cell is infected. Using fluorescent bioimaging and a combination of confocal and electron microscopy we then characterised this interaction in detail, also confirming that human RSV (hRSV) employs the same mechanism. Like hRSV, bRSV viral RNA replication also takes place in the IB, likely meaning these organelles are a functionally conserved feature of orthopneumoviruses.

scholarly journals Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly

2020 ◽  
Vol 6 (14) ◽  
pp. eaaz7095 ◽  
Author(s):  
Serafima Guseva ◽  
Sigrid Milles ◽  
Malene Ringkjøbing Jensen ◽  
Nicola Salvi ◽  
Jean-Philippe Kleman ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Measles Virus ◽  
Weak Interactions ◽  
Intrinsically Disordered ◽  
Infected Cells ◽  
Cellular Compartments ◽  
Liquid Liquid Phase Separation ◽  
Genomic Material

Many viruses are known to form cellular compartments, also called viral factories. Paramyxoviruses, including measles virus, colocalize their proteomic and genomic material in puncta in infected cells. We demonstrate that purified nucleoproteins (N) and phosphoproteins (P) of measles virus form liquid-like membraneless organelles upon mixing in vitro. We identify weak interactions involving intrinsically disordered domains of N and P that are implicated in this process, one of which is essential for phase separation. Fluorescence allows us to follow the modulation of the dynamics of N and P upon droplet formation, while NMR is used to investigate the thermodynamics of this process. RNA colocalizes to droplets, where it triggers assembly of N protomers into nucleocapsid-like particles that encapsidate the RNA. The rate of encapsidation within droplets is enhanced compared to the dilute phase, revealing one of the roles of liquid-liquid phase separation in measles virus replication.

scholarly journals Upon Infection, Cellular WD Repeat-Containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Dzwokai Ma ◽  
Cyril X. George ◽  
Jason L. Nomburg ◽  
Christian K. Pfaller ◽  
Roberto Cattaneo ◽  
...  
Keyword(s):  
Viral Replication ◽  
Inclusion Body ◽  
Virus Replication ◽  
Inclusion Bodies ◽  
Measles Virus ◽  
Rna Replication ◽  
Viral Proteins ◽  
Cellular Protein ◽  
Infected Cells ◽  
Wd Repeat

ABSTRACTReplication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.IMPORTANCEMeasles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.

scholarly journals Viroplasms: Assembly and Functions of Rotavirus Replication Factories

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1349
Author(s):  
Guido Papa ◽  
Alexander Borodavka ◽  
Ulrich Desselberger
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Reverse Genetics ◽  
Intrinsically Disordered Protein ◽  
Structural Proteins ◽  
Virion Assembly ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein–RNA condensates that may be formed via liquid–liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

scholarly journals Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Marie Galloux ◽  
Jennifer Risso-Ballester ◽  
Charles-Adrien Richard ◽  
Jenna Fix ◽  
Marie-Anne Rameix-Welti ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Liquid Phase ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Cytoplasmic Inclusion Bodies ◽  
Syncytial Virus ◽  
P Proteins ◽  
Nucleoprotein N ◽  
Liquid Liquid Phase Separation

ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.

scholarly journals Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

2020 ◽  
Author(s):  
Adriana Savastano ◽  
Alain Ibáñez de Opakua ◽  
Marija Rankovic ◽  
Markus Zweckstetter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Etiologic Agent ◽  
Liquid Phase Separation ◽  
Viral Rna ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

SUMMARYThe etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

scholarly journals A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Shah Kamranur Rahman ◽  
Adeline Kerviel ◽  
Bjorn-Patrick Mohl ◽  
Yao He ◽  
Z. Hong Zhou ◽  
...  
Keyword(s):  
Virus Replication ◽  
Calcium Binding ◽  
Bluetongue Virus ◽  
Inclusion Bodies ◽  
Nonstructural Protein ◽  
Nonstructural Protein 2 ◽  
Terminal Domain ◽  
Ef Hand ◽  
Infected Cells ◽  
Viral Inclusion

ABSTRACT Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways; however, to date, no Ca2+ binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca2+ revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly. IMPORTANCE After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

scholarly journals Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Adriana Savastano ◽  
Alain Ibáñez de Opakua ◽  
Marija Rankovic ◽  
Markus Zweckstetter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Etiologic Agent ◽  
Liquid Phase Separation ◽  
Viral Rna ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

AbstractThe etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid–liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid–liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

scholarly journals Birnaviridae virus factories show features of liquid-liquid phase separation, and are distinct from paracrystalline arrays of virions observed by electron microscopy

2021 ◽  
Author(s):  
Vishwanatha R.A.P. Reddy ◽  
Elle A. Campbell ◽  
Joanna Wells ◽  
Jennifer Simpson ◽  
Salik Nazki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Phase Separation ◽  
Liquid Phase ◽  
Rna Synthesis ◽  
De Novo ◽  
Light And Electron Microscopy ◽  
Region Of Interest ◽  
Liquid Phase Separation ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs co-localized with 5-ethynyl uridine in the presence of actinomycin D, confirming they were the site of de novo RNA synthesis, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 seconds, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower co-localization of the VF GFP signal with the capsid protein VP2 (Manders coefficient (MC) 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs are sites of de novo RNA synthesis, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM.

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