Journal articles: 'Viroplasm'

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Jia, Dongsheng, Nianmei Guo, Hongyan Chen, Fusamichi Akita, Lianhui Xie, Toshihiro Omura, and Taiyun Wei. "Assembly of the viroplasm by viral non-structural protein Pns10 is essential for persistent infection of rice ragged stunt virus in its insect vector." Journal of General Virology 93, no. 10 (October 1, 2012): 2299–309. http://dx.doi.org/10.1099/vir.0.042424-0.

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Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by brown planthopper in a persistent propagative manner. In this study, sequential infection of RRSV in the internal organs of its insect vector after ingestion of virus was investigated by immunofluorescence microscopy. RRSV was first detected in the epithelial cells of the midgut, from where it proceeded to the visceral muscles surrounding the midgut, then throughout the visceral muscles of the midgut and hindgut, and finally into the salivary glands. Viroplasms, the sites of virus replication and assembly of progeny virions, were formed in the midgut epithelium, visceral muscles and salivary glands of infected insects and contained the non-structural protein Pns10 of RRSV, which appeared to be the major constituent of the viroplasms. Viroplasm-like structures formed in non-host insect cells following expression of Pns10 in a baculovirus system, suggesting that the viroplasms observed in RRSV-infected cells were composed basically of Pns10. RNA interference induced by ingestion of dsRNA from the Pns10 gene of RRSV strongly inhibited such viroplasm formation, preventing efficient virus infection and spread in its insect vectors. These results show that Pns10 of RRSV is essential for viroplasm formation and virus replication in the vector insect.

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Taraporewala, Zenobia F., Xiaofang Jiang, Rodrigo Vasquez-Del Carpio, Hariharan Jayaram, B. V. Venkataram Prasad, and John T. Patton. "Structure-Function Analysis of Rotavirus NSP2 Octamer by Using a Novel Complementation System." Journal of Virology 80, no. 16 (August 15, 2006): 7984–94. http://dx.doi.org/10.1128/jvi.00172-06.

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ABSTRACT Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

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Gaunt, Eleanor R., Qifeng Zhang, Winsome Cheung, Michael J. O. Wakelam, Andrew M. L. Lever, and Ulrich Desselberger. "Lipidome analysis of rotavirus-infected cells confirms the close interaction of lipid droplets with viroplasms." Journal of General Virology 94, no. 7 (July 1, 2013): 1576–86. http://dx.doi.org/10.1099/vir.0.049635-0.

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Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms (‘viral factories’) to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11–1.15 g ml−1), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet–viroplasm complexes became enlarged.

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Sun, Liying, Li Xie, Ida Bagus Andika, Zilong Tan, and Jianping Chen. "Non-structural protein P6 encoded by rice black-streaked dwarf virus is recruited to viral inclusion bodies by binding to the viroplasm matrix protein P9-1." Journal of General Virology 94, no. 8 (August 1, 2013): 1908–16. http://dx.doi.org/10.1099/vir.0.051698-0.

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Like other members of the family Reoviridae, rice black-streaked dwarf virus (RBSDV, genus Fijivirus) is thought to replicate and assemble within cytoplasmic viral inclusion bodies, commonly called viroplasms. RBSDV P9-1 is the key protein for the formation of viroplasms, but little is known about the other proteins of the viroplasm or the molecular interactions amongst its components. RBSDV non-structural proteins were screened for their association with P9-1 using a co-immunoprecipitation assay. Only P6 was found to directly interact with P9-1, an interaction that was confirmed by bimolecular fluorescence complementation assay in Spodoptera frugiperda (Sf9) cells. Immunoelectron microscopy showed that P6 and P9-1 co-localized in electron-dense inclusion bodies, indicating that P6 is a constituent of the viroplasm. In addition, non-structural protein P5 also localized to viroplasms and interacted with P6. In Sf9 cells, P6 was diffusely distributed throughout the cytoplasm when expressed alone, but localized to inclusions when co-expressed with P9-1, suggesting that P6 is recruited to viral inclusion bodies by binding to P9-1. P5 localized to the inclusions formed by P9-1 when co-expressed with P6 but did not when P6 was absent, suggesting that P5 is recruited to viroplasms by binding to P6. This study provides a model by which viral non-structural proteins are recruited to RBSDV viroplasms.

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Papa, Guido, Alexander Borodavka, and Ulrich Desselberger. "Viroplasms: Assembly and Functions of Rotavirus Replication Factories." Viruses 13, no. 7 (July 12, 2021): 1349. http://dx.doi.org/10.3390/v13071349.

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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.

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Zhang, Chaozheng, Yueyong Liu, Liyue Liu, Zhiyong Lou, Hongyan Zhang, Hongqin Miao, Xuebo Hu, Yanping Pang, and Bingsheng Qiu. "Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo." Journal of General Virology 89, no. 7 (July 1, 2008): 1770–76. http://dx.doi.org/10.1099/vir.0.2008/000109-0.

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Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, α-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.

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Buchwalter, Rebecca A., Sarah C. Ogden, Sara B. York, Li Sun, Chunfeng Zheng, Christy Hammack, Yichen Cheng, et al. "Coordination of Zika Virus Infection and Viroplasm Organization by Microtubules and Microtubule-Organizing Centers." Cells 10, no. 12 (November 27, 2021): 3335. http://dx.doi.org/10.3390/cells10123335.

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Zika virus (ZIKV) became a global health concern in 2016 due to its links to congenital microcephaly and other birth defects. Flaviviruses, including ZIKV, reorganize the endoplasmic reticulum (ER) to form a viroplasm, a compartment where virus particles are assembled. Microtubules (MTs) and microtubule-organizing centers (MTOCs) coordinate structural and trafficking functions in the cell, and MTs also support replication of flaviviruses. Here we investigated the roles of MTs and the cell’s MTOCs on ZIKV viroplasm organization and virus production. We show that a toroidal-shaped viroplasm forms upon ZIKV infection, and MTs are organized at the viroplasm core and surrounding the viroplasm. We show that MTs are necessary for viroplasm organization and impact infectious virus production. In addition, the centrosome and the Golgi MTOC are closely associated with the viroplasm, and the centrosome coordinates the organization of the ZIKV viroplasm toroidal structure. Surprisingly, viroplasm formation and virus production are not significantly impaired when infected cells have no centrosomes and impaired Golgi MTOC, and we show that MTs are anchored to the viroplasm surface in these cells. We propose that the viroplasm is a site of MT organization, and the MTs organized at the viroplasm are sufficient for efficient virus production.

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Criglar, Jeanette M., Ramakrishnan Anish, Liya Hu, Sue E. Crawford, Banumathi Sankaran, B. V. Venkataram Prasad, and Mary K. Estes. "Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories." Proceedings of the National Academy of Sciences 115, no. 51 (December 3, 2018): E12015—E12023. http://dx.doi.org/10.1073/pnas.1717944115.

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The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.

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Ngo, Thu Ha, Richard Webb, Kathleen S. Crew, Megan E. Vance, John E. Thomas, and Andrew D. W. Geering. "Identification of putative viroplasms within banana cells infected by banana streak MY virus." Journal of General Virology 101, no. 12 (December 1, 2020): 1305–12. http://dx.doi.org/10.1099/jgv.0.001498.

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The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.

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Cheung, Winsome, Michael Gill, Alessandro Esposito, Clemens F. Kaminski, Nathalie Courousse, Serge Chwetzoff, Germain Trugnan, Nandita Keshavan, Andrew Lever, and Ulrich Desselberger. "Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication." Journal of Virology 84, no. 13 (March 24, 2010): 6782–98. http://dx.doi.org/10.1128/jvi.01757-09.

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ABSTRACT Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

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Campagna, Michela, Catherine Eichwald, Fulvia Vascotto, and Oscar R. Burrone. "RNA interference of rotavirus segment 11 mRNA reveals the essential role of NSP5 in the virus replicative cycle." Journal of General Virology 86, no. 5 (May 1, 2005): 1481–87. http://dx.doi.org/10.1099/vir.0.80598-0.

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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.

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Martins, Claudia R. F., Jennifer A. Johnson, Diane M. Lawrence, Tae-Jin Choi, Anna-Maria Pisi, Sara L. Tobin, Denise Lapidus, et al. "Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins." Journal of Virology 72, no. 7 (July 1, 1998): 5669–79. http://dx.doi.org/10.1128/jvi.72.7.5669-5679.1998.

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ABSTRACT We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of β-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

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Deng, Min, Jennifer N. Bragg, Steven Ruzin, Denise Schichnes, David King, Michael M. Goodin, and Andrew O. Jackson. "Role of the Sonchus Yellow Net Virus N Protein in Formation of Nuclear Viroplasms." Journal of Virology 81, no. 10 (March 7, 2007): 5362–74. http://dx.doi.org/10.1128/jvi.02349-06.

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ABSTRACT Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells and that homologous interactions mediated by a helix-loop-helix region near the amino terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N- and P-protein interactions that are required for N and P colocalization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N-N and N-P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin α homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin α binding. The P protein did not bind to the importin α homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection, the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N-protein interactions, and that P protein targeting to the viroplasm requires N-P protein interactions that occur after N and P protein import into the nucleus.

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Campagna, M., L. Marcos-Villar, F. Arnoldi, C. F. de la Cruz-Herrera, P. Gallego, J. Gonzalez-Santamaria, D. Gonzalez, et al. "Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation." Journal of Virology 87, no. 2 (October 31, 2012): 807–17. http://dx.doi.org/10.1128/jvi.01578-12.

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Silvestri, Lynn S., Zenobia F. Taraporewala, and John T. Patton. "Rotavirus Replication: Plus-Sense Templates for Double-Stranded RNA Synthesis Are Made in Viroplasms." Journal of Virology 78, no. 14 (July 15, 2004): 7763–74. http://dx.doi.org/10.1128/jvi.78.14.7763-7774.2004.

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ABSTRACT Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.

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Kumar, Mukesh, Hariharan Jayaram, Rodrigo Vasquez-Del Carpio, Xiaofang Jiang, Zenobia F. Taraporewala, Raymond H. Jacobson, John T. Patton, and B. V. Venkataram Prasad. "Crystallographic and Biochemical Analysis of Rotavirus NSP2 with Nucleotides Reveals a Nucleoside Diphosphate Kinase-Like Activity." Journal of Virology 81, no. 22 (September 5, 2007): 12272–84. http://dx.doi.org/10.1128/jvi.00984-07.

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ABSTRACT Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.

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Szajner, Patricia, Andrea S. Weisberg, Elizabeth J. Wolffe, and Bernard Moss. "Vaccinia Virus A30L Protein Is Required for Association of Viral Membranes with Dense Viroplasm To Form Immature Virions." Journal of Virology 75, no. 13 (July 1, 2001): 5752–61. http://dx.doi.org/10.1128/jvi.75.13.5752-5761.2001.

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ABSTRACT The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.

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Silvestri, Lynn S., M. Alejandra Tortorici, Rodrigo Vasquez-Del Carpio, and John T. Patton. "Rotavirus Glycoprotein NSP4 Is a Modulator of Viral Transcription in the Infected Cell." Journal of Virology 79, no. 24 (December 15, 2005): 15165–74. http://dx.doi.org/10.1128/jvi.79.24.15165-15174.2005.

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ABSTRACT The outer shell of the rotavirus triple-layered virion is lost during cell entry, yielding a double-layered particle (DLP) that directs synthesis of viral plus-strand RNAs. The plus-strand RNAs act as templates for synthesis of the segmented double-stranded RNA (dsRNA) genome in viral inclusion bodies (viroplasms). The viral endoplasmic reticulum (ER)-resident glycoprotein NSP4 recruits progeny DLPs formed in viroplasms to the ER, where the particles are converted to triple-layered particles (TLPs) via budding. In this study, we have used short interfering RNAs to probe the role of NSP4 in the viral life cycle. Our analysis showed that knockdown of NSP4 expression had no marked effect on the expression of other viral proteins or on the replication of the dsRNA genome segments. However, NSP4 loss of function suppressed viroplasm maturation and caused a maldistribution of nonstructural and structural proteins that normally accumulate in viroplasms. NSP4 loss of function also inhibited formation of packaged virus particles, instead inducing the accumulation of empty particles. Most significant was the observation that NSP4 knockdown led to dramatically increased levels of viral transcription late in the infection cycle. These findings point to a multifaceted role for NSP4 in virus replication, including influencing the development of viroplasms, linking genome packaging with particle assembly, and acting as a modulator of viral transcription. By recruiting transcriptionally active or potentially active DLPs to the ER for conversion to quiescent TLPs, NSP4 acts as a feedback inhibitor down-regulating viral transcription when adequate levels of plus-strand RNAs are available to allow for productive infection.

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Akita, Fusamichi, Naoyuki Miyazaki, Hiroyuki Hibino, Takumi Shimizu, Akifumi Higashiura, Tamaki Uehara-Ichiki, Takahide Sasaya, et al. "Viroplasm matrix protein Pns9 from rice gall dwarf virus forms an octameric cylindrical structure." Journal of General Virology 92, no. 9 (September 1, 2011): 2214–21. http://dx.doi.org/10.1099/vir.0.032524-0.

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The non-structural Pns9 protein of rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. Immunofluorescence and immunoelectron microscopy of RGDV-infected vector cells in monolayers, using antibodies against Pns9 of RGDV and expression of Pns9 in Spodoptera frugiperda cells, demonstrated that Pns9 is the minimal viral factor necessary for formation of viroplasm inclusion during infection by RGDV. When Pns9 in solution was observed under a conventional electron microscope, it appeared as ring-like aggregates of approximately 100 Å in diameter. Cryo-electron microscopic analysis of these aggregates revealed cylinders of octameric Pns9, whose dimensions were similar to those observed under the conventional electron microscope. Octamerization of Pns9 in solution was confirmed by the results of size-exclusion chromatography. Among proteins of viruses that belong to the family Reoviridae whose three-dimensional structures are available, a matrix protein of the viroplasm of rotavirus, NSP2, forms similar octamers, an observation that suggests similar roles for Pns9 and NSP2 in morphogenesis in animal-infecting and in plant-infecting reoviruses.

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De Lorenzo, G., C. Eichwald, E. M. Schraner, V. Nicolin, R. Bortul, M. Mano, O. R. Burrone, and F. Arnoldi. "Production of in vivo-biotinylated rotavirus particles." Journal of General Virology 93, no. 7 (July 1, 2012): 1474–82. http://dx.doi.org/10.1099/vir.0.040089-0.

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Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the in vivo incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. In vivo biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6–BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6–BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.

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Eichwald, Catherine, Fulvia Vascotto, Elsa Fabbretti, and Oscar R. Burrone. "Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains." Journal of Virology 76, no. 7 (April 1, 2002): 3461–70. http://dx.doi.org/10.1128/jvi.76.7.3461-3470.2002.

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ABSTRACT 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 (Ser153, Ser155, Ser163, and Ser165) 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.

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Wu, Jianyan, Jia Li, Xiang Mao, Weiwu Wang, Zhaobang Cheng, Yijun Zhou, Xueping Zhou, and Xiaorong Tao. "Viroplasm Protein P9-1 ofRice Black-Streaked Dwarf VirusPreferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site." Journal of Virology 87, no. 23 (September 25, 2013): 12885–99. http://dx.doi.org/10.1128/jvi.02264-13.

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The P9-1 protein ofRice black-streaked dwarf virus(RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed forin vitrobinding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

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Arnoldi, F., M. Campagna, C. Eichwald, U. Desselberger, and O. R. Burrone. "Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2." Journal of Virology 81, no. 5 (December 20, 2006): 2128–37. http://dx.doi.org/10.1128/jvi.01494-06.

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ABSTRACT Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.

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López, Tomás, Margarito Rojas, Camilo Ayala-Bretón, Susana López, and Carlos F. Arias. "Reduced expression of the rotavirus NSP5 gene has a pleiotropic effect on virus replication." Journal of General Virology 86, no. 6 (June 1, 2005): 1609–17. http://dx.doi.org/10.1099/vir.0.80827-0.

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Rotavirus RRV gene 11 encodes two non-structural proteins, NSP5 and NSP6. NSP5 is a phosphorylated non-structural protein that binds single- and double-stranded RNA in a non-specific manner. Transient expression of this protein in uninfected cells has provided evidence for its participation in the formation of electron-dense cytoplasmic structures, known as viroplasms, which are thought to be key structures for the replication of the virus. NSP6 is a protein of unknown function that seems not to be essential for virus replication in cell culture. To study the function of NSP5 in the context of a viral infection, the expression of RRV gene 11 was silenced by RNA interference. Reduction in the synthesis of NSP5, as shown by immunoblot and immunofluorescence assays, correlated with a reduction in the number and size of viroplasms and with an altered intracellular distribution of other viroplasm-associated proteins. Silencing of gene 11 also resulted in a reduced synthesis of viral RNA(+) and double-stranded RNA and of all viral proteins, as well as in a decreased production of infectious virus. A similar phenotype was observed when the NSP5 coding gene of the lapine rotavirus strain Alabama was silenced. The fact that the NSP5 gene of rotavirus Alabama lacks the AUG initiator codon for a complete NSP6 protein, suggests that the described phenotype in gene 11-silenced cells is mostly due to the absence of NSP5. The data presented in this work suggest that NSP5 is a key protein during the replication cycle of rotaviruses.

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Wang, Qian, Tao Tao, Yanjing Zhang, Wenqi Wu, Dawei Li, Jialin Yu, and Chenggui Han. "Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1." Virology Journal 8, no. 1 (2011): 24. http://dx.doi.org/10.1186/1743-422x-8-24.

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Risco, Cristina, Juan R. Rodríguez, Carmen López-Iglesias, José L. Carrascosa, Mariano Esteban, and Dolores Rodríguez. "Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly." Journal of Virology 76, no. 4 (February 15, 2002): 1839–55. http://dx.doi.org/10.1128/jvi.76.4.1839-1855.2002.

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ABSTRACT Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements.

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Szajner, Patricia, Andrea S. Weisberg, and Bernard Moss. "Unique Temperature-Sensitive Defect in Vaccinia Virus Morphogenesis Maps to a Single Nucleotide Substitution in the A30L Gene." Journal of Virology 75, no. 22 (November 15, 2001): 11222–26. http://dx.doi.org/10.1128/jvi.75.22.11222-11226.2001.

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ABSTRACT Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.

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Takano, Yoshihito, Yuji Tomaru, and Keizo Nagasaki. "Visualization of a Dinoflagellate-Infecting Virus HcDNAV and Its Infection Process." Viruses 10, no. 10 (October 11, 2018): 554. http://dx.doi.org/10.3390/v10100554.

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HcDNAV (a type species of Genus Dinodnavirus) is a large double-stranded DNA virus, which lytically infects the bloom-forming marine microalga Heterocapsa circularisquama Horiguchi (Dinophyceae). In the present study, detailed observation of the HcDNAV particle and its infection process was conducted via field emission scanning electron microscopy (FE-SEM) and epifluorescence microscopy (EFM). Each five-fold vertex of the icosahedral virion was decorated with a protrusion, which may be related to the entry process of HcDNAV into the host. The transverse groove of host cells is proposed to be the main virus entry site. A visible DAPI-stained region, which is considered to be the viroplasm (virus factory), appeared in close proximity to the host nucleus at 11 h post infection (hpi); the putative viral DAPI signal was remarkably enlarged at 11–30 hpi. It was kidney-shaped at 13–15 hpi, horseshoe-shaped at 20 hpi, doughnut-shaped at 30 hpi, and changed into a three-dimensionally complicated shape at 51–53 hpi, by which time most parts of the host cell were occupied by the putative viral DAPI signal. While the virions were within the viroplasm, they were easily distinguishable by their vertex protrusions by FE-SEM.

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Fang, Xiao-Dong, Teng Yan, Qiang Gao, Qing Cao, Dong-Min Gao, Wen-Ya Xu, Zhen-Jia Zhang, Zhi-Hang Ding, and Xian-Bing Wang. "A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis." Journal of Experimental Botany 70, no. 15 (April 25, 2019): 4049–62. http://dx.doi.org/10.1093/jxb/erz195.

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AbstractAs obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43–55) and the remaining region (aa 56–295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

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Carreño-Torres, José J., Michelle Gutiérrez, Carlos F. Arias, Susana López, and Pavel Isa. "Characterization of viroplasm formation during the early stages of rotavirus infection." Virology Journal 7, no. 1 (2010): 350. http://dx.doi.org/10.1186/1743-422x-7-350.

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Netherton, Christopher L., and Tom Wileman. "Virus factories, double membrane vesicles and viroplasm generated in animal cells." Current Opinion in Virology 1, no. 5 (November 2011): 381–87. http://dx.doi.org/10.1016/j.coviro.2011.09.008.

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Martin, Davy, Mariela Duarte, Jean Lepault, and Didier Poncet. "Sequestration of Free Tubulin Molecules by the Viral Protein NSP2 Induces Microtubule Depolymerization during Rotavirus Infection." Journal of Virology 84, no. 5 (December 23, 2009): 2522–32. http://dx.doi.org/10.1128/jvi.01883-09.

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ABSTRACT Microtubules, components of the cell cytoskeleton, play a central role in cellular trafficking. Here we show that rotavirus infection leads to a remodeling of the microtubule network together with the formation of tubulin granules. While most microtubules surrounding the nucleus depolymerize, others appear packed at the cell periphery. In microtubule depolymerization areas, tubulin granules are observed; they colocalize with viroplasms, viral compartments formed by interactions between rotavirus proteins NSP2 and NSP5. With purified proteins, we show that tubulin directly interacts in vitro with NSP2 but not with NSP5. The binding of NSP2 to tubulin is independent of its phosphatase activity. The comparison of three-dimensional (3-D) reconstructions of NSP2 octamers alone or associated with tubulin reveals electron densities in the positively charged grooves of NSP2 that we attribute to tubulin. Site-directed mutagenesis of NSP2 and competition assays between RNA and tubulin for NSP2 binding confirm that tubulin binds to these charged grooves of NSP2. Although the tubulin position within NSP2 grooves cannot be precisely determined, the tubulin C-terminal H12 α-helix could be involved in the interaction. NSP2 overexpression and rotavirus infection produce similar effects on the microtubule network. NSP2 depolymerizes microtubules and leads to tubulin granule formation. Our results demonstrate that tubulin is a viroplasm component and reveal an original mechanism. Tubulin sequestration by NSP2 induces microtubule depolymerization. This depolymerization probably reroutes the cell machinery by inhibiting trafficking and functions potentially involved in defenses to viral infections.

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Rodríguez, D., D. López-Abella, and J. R. Díaz-Ruiz. "An electron microscopic study of cauliflower mosaic virus-induced viroplasms: Unusual structures within the viroplasm matrix with possible functional significance in the viral replication cycle." Journal of Ultrastructure and Molecular Structure Research 100, no. 2 (August 1988): 118–25. http://dx.doi.org/10.1016/0889-1605(88)90019-5.

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34

Eichwald, Catherine, Francesca Arnoldi, Andrea S. Laimbacher, Elisabeth M. Schraner, Cornel Fraefel, Peter Wild, Oscar R. Burrone, and Mathias Ackermann. "Rotavirus Viroplasm Fusion and Perinuclear Localization Are Dynamic Processes Requiring Stabilized Microtubules." PLoS ONE 7, no. 10 (October 23, 2012): e47947. http://dx.doi.org/10.1371/journal.pone.0047947.

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Criglar, Jeanette M., Sue E. Crawford, and Mary K. Estes. "Plasmid-based reverse genetics for probing phosphorylation-dependent viroplasm formation in rotaviruses." Virus Research 291 (January 2021): 198193. http://dx.doi.org/10.1016/j.virusres.2020.198193.

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Eichwald, Catherine, José Francisco Rodriguez, and Oscar R. Burrone. "Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation." Journal of General Virology 85, no. 3 (March 1, 2004): 625–34. http://dx.doi.org/10.1099/vir.0.19611-0.

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37

Maruri-Avidal, Liliana, Andrea S. Weisberg, Himani Bisht, and Bernard Moss. "Analysis of Viral Membranes Formed in Cells Infected by a Vaccinia Virus L2-Deletion Mutant Suggests Their Origin from the Endoplasmic Reticulum." Journal of Virology 87, no. 3 (November 28, 2012): 1861–71. http://dx.doi.org/10.1128/jvi.02779-12.

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ABSTRACTAssembly of the poxvirus immature virion (IV) membrane is a poorly understood event that occurs within the cytoplasm. At least eight viral proteins participate in formation of the viral membrane. Of these, A14, A17, and D13 are structural components whereas A6, A11, F10, H7, and L2 participate in membrane biogenesis. L2, the object of this study, is conserved in all chordopoxviruses, expressed early in infection, and associated with the endoplasmic reticulum (ER) throughout the cell and at the edges of crescent-shaped IV precursors. Previous studies with an inducible L2 mutant revealed abortive formation of the crescent membrane. However, possible low-level L2 synthesis under nonpermissive conditions led to ambiguity in interpretation. Here, we constructed a cell line that expresses L2, which allowed the creation of an L2-deletion mutant. In noncomplementing cells, replication was aborted prior to formation of mature virions and two types of aberrant structures were recognized. One consisted of short crescents, at the surface of dense masses of viroplasm, which were labeled with antibodies to the A11, A14, A17, and D13 proteins. The other structure consisted of “empty” IV-like membranes, also labeled with antibodies to the viral proteins, which appeared to be derived from adjacent calnexin-containing ER. A subset of 25 proteins examined, exemplified by components of the entry-fusion complex, were greatly diminished in amount. The primary role of L2 may be to recruit ER and modulate its transformation to viral membranes in juxtaposition with the viroplasm, simultaneously preventing the degradation of viral proteins dependent on viral membranes for stability.

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Sen, Adrish, Darin Agresti, and Erich R. Mackow. "Hyperphosphorylation of the Rotavirus NSP5 Protein Is Independent of Serine 67 or NSP2, and the Intrinsic Insolubility of NSP5 Is Regulated by Cellular Phosphatases." Journal of Virology 80, no. 4 (February 15, 2006): 1807–16. http://dx.doi.org/10.1128/jvi.80.4.1807-1816.2006.

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ABSTRACT The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32- to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.

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Goodin, Michael M., Romit Chakrabarty, Sharon Yelton, Kathleen Martin, Anthony Clark, and Robert Brooks. "Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant-adapted rhabdovirus." Journal of General Virology 88, no. 6 (June 1, 2007): 1810–20. http://dx.doi.org/10.1099/vir.0.82698-0.

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Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells.

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Fabbretti, E., I. Afrikanova, F. Vascotto, and O. R. Burrone. "Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo." Journal of General Virology 80, no. 2 (February 1, 1999): 333–39. http://dx.doi.org/10.1099/0022-1317-80-2-333.

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41

Akita, F., A. Higashiura, T. Shimizu, Y. Pu, M. Suzuki, T. Uehara-Ichiki, T. Sasaya, T. Tsukihara, A. Nakagawa, and T. Omura. "Crystal structure of the viroplasm matrix protein P9-1 of Rice black streaked dwarf virus." Acta Crystallographica Section A Foundations of Crystallography 67, a1 (August 22, 2011): C412. http://dx.doi.org/10.1107/s0108767311089665.

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42

Nilsson, Mikael, Carl-Henrik von Bonsdorff, Katharzyna Weclewicz, Jean Cohen, and Lennart Svensson. "Assembly of Viroplasm and Virus-like Particles of Rotavirus by a Semliki Forest Virus Replicon." Virology 242, no. 2 (March 1998): 255–65. http://dx.doi.org/10.1006/viro.1997.8987.

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43

Desser, Sherwin S. "Ultrastructural observations on an icosahedral cytoplasmic virus in leukocytes of frogs from Algonquin Park, Ontario." Canadian Journal of Zoology 70, no. 4 (April 1, 1992): 833–36. http://dx.doi.org/10.1139/z92-118.

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An icosahedral virus was observed in mononuclear leukocytes and heterophils of a mink frog (Rana septentrionalis) and a green frog (Rana clamitans). Infected cells were grossly distorted, often containing a prominent juxtanuclear viroplasm in which nucleocapsids in various stages of assembly occurred. Viral particles were also observed occasionally within the nucleus of some cells. Mature nucleocapsids were usually hexagonal in profile, measuring about 131 ± 9.2 nm in diameter (n = 10) and were arranged in paracrystalline arrays. Individual viral particles were observed at the periphery of infected cells apparently budding from the plasma membrane. These observations extend both the geographic and host ranges of frog leukocytic virus which could serve as a useful model, not only for the study of viral function, but also for its effect on the immune system of the anuran host.

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Wei, Taiyun, Hiroyuki Hibino, and Toshihiro Omura. "Rice dwarf virus is engulfed into and released via vesicular compartments in cultured insect vector cells." Journal of General Virology 89, no. 11 (November 1, 2008): 2915–20. http://dx.doi.org/10.1099/vir.0.2008/002063-0.

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Vector insect cells infected with Rice dwarf virus had vesicular compartments containing viral particles located adjacent to the viroplasm when examined by transmission electron and confocal microscopy. Such compartments were often at the periphery of infected cells. Inhibitors of vesicular transport, brefeldin A and monensin, and an inhibitor of myosin motor activity, butanedione monoxime, abolished the formation of such vesicles and prevented the release of viral particles from infected cells without significant effects on virus multiplication. Furthermore, the actin-depolymerizing drug, cytochalasin D, inhibited the formation of actin filaments without significantly interfering with formation of vesicular compartments and the release of viruses from treated cells. These results together revealed intracellular vesicular compartments as a mode for viral transport in and release from insect vector cells infected with a plant-infecting reovirus.

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Akita, F., A. Higashiura, T. Shimizu, Y. Pu, M. Suzuki, T. Uehara-Ichiki, T. Sasaya, et al. "Crystallographic Analysis Reveals Octamerization of Viroplasm Matrix Protein P9-1 of Rice Black Streaked Dwarf Virus." Journal of Virology 86, no. 2 (November 9, 2011): 746–56. http://dx.doi.org/10.1128/jvi.00826-11.

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Touris-Otero, Fernando, José Martı́nez-Costas, Vikram N. Vakharia, and Javier Benavente. "Avian reovirus nonstructural protein μNS forms viroplasm-like inclusions and recruits protein σNS to these structures." Virology 319, no. 1 (February 2004): 94–106. http://dx.doi.org/10.1016/j.virol.2003.10.034.

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47

Sen, Adrish, Nandini Sen, and Erich R. Mackow. "The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain." Journal of Virology 81, no. 21 (August 15, 2007): 11758–67. http://dx.doi.org/10.1128/jvi.01124-07.

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

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Criglar, J. M., L. Hu, S. E. Crawford, J. M. Hyser, J. R. Broughman, B. V. V. Prasad, and M. K. Estes. "A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly." Journal of Virology 88, no. 2 (November 6, 2013): 786–98. http://dx.doi.org/10.1128/jvi.03022-13.

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Wei, T., A. Kikuchi, N. Suzuki, T. Shimizu, K. Hagiwara, H. Chen, and T. Omura. "Pns4 of rice dwarf virus is a phosphoprotein, is localized around the viroplasm matrix, and forms minitubules." Archives of Virology 151, no. 9 (April 13, 2006): 1701–12. http://dx.doi.org/10.1007/s00705-006-0757-4.

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La Frazia, S., A. Ciucci, F. Arnoldi, M. Coira, P. Gianferretti, M. Angelini, G. Belardo, O. R. Burrone, J. F. Rossignol, and M. G. Santoro. "Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation." Journal of Virology 87, no. 20 (August 7, 2013): 11096–106. http://dx.doi.org/10.1128/jvi.01213-13.

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Journal articles on the topic 'Viroplasm'

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