Journal articles: 'Viroplasmic Proteins'

1

Berois, Mabel, Catherine Sapin, Inge Erk, Didier Poncet, and Jean Cohen. "Rotavirus Nonstructural Protein NSP5 Interacts with Major Core Protein VP2." Journal of Virology 77, no. 3 (February 1, 2003): 1757–63. http://dx.doi.org/10.1128/jvi.77.3.1757-1763.2003.

Full text

Abstract:

ABSTRACT Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.

APA, Harvard, Vancouver, ISO, and other styles

2

Contin, R., F. Arnoldi, M. Campagna, and O. R. Burrone. "Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins." Journal of General Virology 91, no. 7 (March 3, 2010): 1782–93. http://dx.doi.org/10.1099/vir.0.019133-0.

Full text

APA, Harvard, Vancouver, ISO, and other styles

3

González, R. A., R. Espinosa, P. Romero, S. López, and C. F. Arias. "Relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells." Archives of Virology 145, no. 9 (September 2000): 1963–73. http://dx.doi.org/10.1007/s007050070069.

Full text

APA, Harvard, Vancouver, ISO, and other styles

4

Wang, Dongmei, Xin Xie, Di Gao, Kai Chen, Zhuo Chen, Linhong Jin, Xiangyang Li, and Baoan Song. "Dufulin Intervenes the Viroplasmic Proteins as the Mechanism of Action against Southern Rice Black-Streaked Dwarf Virus." Journal of Agricultural and Food Chemistry 67, no. 41 (September 19, 2019): 11380–87. http://dx.doi.org/10.1021/acs.jafc.9b05793.

Full text

APA, Harvard, Vancouver, ISO, and other styles

5

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

6

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

7

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

8

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

9

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

10

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

11

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

12

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

13

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

14

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

15

Heljasvaara, Ritva, Dolores Rodrı́guez, Cristina Risco, José L. Carrascosa, Mariano Esteban, and Juan Ramón Rodrı́guez. "The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles." Journal of Virology 75, no. 13 (July 1, 2001): 5778–95. http://dx.doi.org/10.1128/jvi.75.13.5778-5795.2001.

Full text

Abstract:

ABSTRACT The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of theEscherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

APA, Harvard, Vancouver, ISO, and other styles

16

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

17

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

18

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

19

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

20

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

21

Wei, Taiyun, Takumi Shimizu, Kyoji Hagiwara, Akira Kikuchi, Yusuke Moriyasu, Nobuhiro Suzuki, Hongyan Chen, and Toshihiro Omura. "Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes." Journal of General Virology 87, no. 2 (February 1, 2006): 429–38. http://dx.doi.org/10.1099/vir.0.81425-0.

Full text

Abstract:

Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of members of the family Reoviridae. Immunocytochemical and biochemical analyses were carried out to characterize the poorly understood viroplasms of the phytoreovirus Rice dwarf virus (RDV). Within 6 h of inoculation of cells, viroplasms, namely discrete cytoplasmic inclusions, were formed that contained the non-structural proteins Pns6, Pns11 and Pns12 of RDV, which appeared to be the constituents of the inclusions. Formation of similar inclusions in non-host insect cells upon expression of Pns12 in a baculovirus system and the association of molecules of Pns12 in vitro suggested that the inclusions observed in RDV-infected cells were composed basically of Pns12. Core proteins P1, P3, P5 and P7 and core virus particles were identified in the interior region of the inclusions. In contrast, accumulation of the outer capsid proteins P2, P8 and P9 and of intact virus particles was evident in the peripheral regions of the inclusions. These observations suggest that core particles were constructed inside the inclusions, whereas outer capsid proteins were assembled at the periphery of the inclusions. Viral inclusions were shown to be the sites of viral RNA synthesis by labelling infected cells with 5-bromouridine 5′-triphosphate. The number of viroplasms decreased with time post-inoculation as their sizes increased, suggesting that inclusions might fuse with one another during the virus-propagation process. Our results are consistent with a model, proposed for vertebrate reoviruses, in which viroplasms play a pivotal role in virus assembly.

APA, Harvard, Vancouver, ISO, and other styles

22

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

23

Campagna, Michela, Mauricio Budini, Francesca Arnoldi, Ulrich Desselberger, Jorge E. Allende, and Oscar R. Burrone. "Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1α is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication." Journal of General Virology 88, no. 10 (October 1, 2007): 2800–2810. http://dx.doi.org/10.1099/vir.0.82922-0.

Full text

Abstract:

The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1α is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1α is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1α was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1α is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection.

APA, Harvard, Vancouver, ISO, and other styles

24

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.

Full text

APA, Harvard, Vancouver, ISO, and other styles

25

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

26

Wei, Taiyun, Tamaki Uehara-Ichiki, Naoyuki Miyazaki, Hiroyuki Hibino, Kenji Iwasaki, and Toshihiro Omura. "Association of Rice Gall Dwarf Virus with Microtubules Is Necessary for Viral Release from Cultured Insect Vector Cells." Journal of Virology 83, no. 20 (July 29, 2009): 10830–35. http://dx.doi.org/10.1128/jvi.01067-09.

Full text

Abstract:

ABSTRACT Vector insect cells infected with Rice gall dwarf virus, a member of the family Reoviridae, contained the virus-associated microtubules adjacent to the viroplasms, as revealed by transmission electron, electron tomographic, and confocal microscopy. The viroplasms, putative sites of viral replication, contained the nonstructural viral proteins Pns7 and Pns12, as well as core protein P5, of the virus. Microtubule-depolymerizing drugs suppressed the association of viral particles with microtubules and prevented the release of viruses from cells without significantly affecting viral multiplication. Thus, microtubules appear to mediate viral transport within and release of viruses from infected vector cells.

APA, Harvard, Vancouver, ISO, and other styles

27

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

28

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

29

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

30

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.

Full text

APA, Harvard, Vancouver, ISO, and other styles

31

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

32

Berkova, Z., S. E. Crawford, G. Trugnan, T. Yoshimori, A. P. Morris, and M. K. Estes. "Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms." Journal of Virology 80, no. 12 (June 15, 2006): 6061–71. http://dx.doi.org/10.1128/jvi.02167-05.

Full text

Abstract:

ABSTRACT Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

APA, Harvard, Vancouver, ISO, and other styles

33

Buck, Kenneth W. "Replication of tobacco mosaic virus RNA." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 354, no. 1383 (March 29, 1999): 613–27. http://dx.doi.org/10.1098/rstb.1999.0413.

Full text

Abstract:

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative–strand RNA using the genomic positive–strand RNA as a template, followed by the synthesis of positive–strand RNA on the negative–strand RNA templates. Intermediates of replication isolated from infected cells include completely double–stranded RNA (replicative form) and partly double–stranded and partly single–stranded RNA (replicative intermediate), but it is not known whether these structures are double–stranded or largely single–stranded in vivo . The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic–length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus–encoded 126–kDa protein, which has amino–acid sequence motifs typical of methyltransferases and helicases, and the 183–kDa protein, which has additional motifs characteristic of RNA–dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA–binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm–1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce ‘X bodies’. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single–stranded RNA.

APA, Harvard, Vancouver, ISO, and other styles

34

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

35

Kondo, H., S. Chiba, I. B. Andika, K. Maruyama, T. Tamada, and N. Suzuki. "Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection." Journal of Virology 87, no. 13 (April 24, 2013): 7423–34. http://dx.doi.org/10.1128/jvi.00270-13.

Full text

APA, Harvard, Vancouver, ISO, and other styles

36

Szécsi, Judit, Xin Shun Ding, Chae Oh Lim, Mohammed Bendahmane, Moo Je Cho, Richard S. Nelson, and Roger N. Beachy. "Development of Tobacco Mosaic Virus Infection Sites in Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 12, no. 2 (February 1999): 143–52. http://dx.doi.org/10.1094/mpmi.1999.12.2.143.

Full text

Abstract:

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 183 kDa replicase proteins accumulated in paired cytoplasmic bodies that formed often on opposite sides of adjacent cell walls containing plasmodesmata. In the dimly fluorescent center of the rings the 126 kDa and 183 kDa proteins, but not the MP:GFP, were localized in unpaired cytoplasmic bodies containing ropelike, fibrillar structures. The paired bodies were similar to previously described viroplasms, while the unpaired bodies were similar to X-bodies. These data indicate that the accumulation of proteins expressed from different sgps of TMV has a specific spatial and temporal pattern in planta. In addition, the cytoplasmic bodies containing the 126 kDa and 183 kDa proteins are dynamic entities whose protein content and subcellular location change during infection.

APA, Harvard, Vancouver, ISO, and other styles

37

Llauger, Gabriela, Luis Alejandro de Haro, Victoria Alfonso, and Mariana del Vas. "Interaction of Mal de Río Cuarto virus ( Fijivirus genus) proteins and identification of putative factors determining viroplasm formation and decay." Virus Research 230 (February 2017): 19–28. http://dx.doi.org/10.1016/j.virusres.2017.01.002.

Full text

APA, Harvard, Vancouver, ISO, and other styles

38

Mohan, K. V. K., J. Muller, and C. D. Atreya. "The N- and C-Terminal Regions of Rotavirus NSP5 Are the Critical Determinants for the Formation of Viroplasm-Like Structures Independent of NSP2." Journal of Virology 77, no. 22 (November 15, 2003): 12184–92. http://dx.doi.org/10.1128/jvi.77.22.12184-12192.2003.

Full text

Abstract:

ABSTRACT Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.

APA, Harvard, Vancouver, ISO, and other styles

39

Barbosa, Bruna Rocha Passos, Nara Thiers Cacciatori Galleti Bernardes, Laila Andreia Rodrigues Beserra, and Fábio Gregori. "Molecular Characterization of the Porcine Group A Rotavirus NSP2 and NSP5/6 Genes from São Paulo State, Brazil, in 2011/12." Scientific World Journal 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/241686.

Full text

Abstract:

Rotaviruses are responsible for the acute diarrhea in various mammalian and avian species. The nonstructural proteins NSP2 and NSP5 are involved in the rotavirus replication and the formation of viroplasm, cytoplasmic inclusion bodies within which new viral particles morphogenesis and viral RNA replication occur. There are few studies on the genetic diversity of those proteins; thus this study aims at characterizing the diversity of rotavirus based on NSP2 and NSP5 genes in rotaviruses circulating in Brazilian pig farms. For this purpose, 63 fecal samples from pig farms located in six different cities in the São Paulo State, Brazil, were screened by nested RT-PCR. Seven strains had the partial nucleotide sequencing for NSP2, whereas in six, the total sequencing for NSP5. All were characterized as genotype H1 and N1. The nucleotide identity of NSP2 genes ranged from 100% to 86.4% and the amino acid identity from 100% to 91.5%. For NSP5, the nucleotide identity was from 100% to 95.1% and the amino acid identity from 100% to 97.4%. It is concluded that the genotypes of the strains circulating in the region of study are in agreement with those reported in the literature for swine and that there is the possibility of interaction between human and animal rotaviruses.

APA, Harvard, Vancouver, ISO, and other styles

40

Songbai, Zhang, Du Zhenguo, Yang Liang, Yuan Zhengjie, Wu Kangcheng, Li Guangpu, Wu Zujian, and Xie Lianhui. "Identification and characterization of the interaction between viroplasm-associated proteins from two different plant-infecting reoviruses and eEF-1A of rice." Archives of Virology 158, no. 10 (April 19, 2013): 2031–39. http://dx.doi.org/10.1007/s00705-013-1703-x.

Full text

APA, Harvard, Vancouver, ISO, and other styles

41

Szajner, Patricia, Howard Jaffe, Andrea S. Weisberg, and Bernard Moss. "Vaccinia Virus G7L Protein Interacts with the A30L Protein and Is Required for Association of Viral Membranes with Dense Viroplasm To Form Immature Virions." Journal of Virology 77, no. 6 (March 15, 2003): 3418–29. http://dx.doi.org/10.1128/jvi.77.6.3418-3429.2003.

Full text

Abstract:

ABSTRACT The vaccinia virus A30L protein is required for the association of electron-dense, granular, proteinaceous material with the concave surfaces of crescent membranes, an early step in viral morphogenesis. For the identification of additional proteins involved in this process, we used an antibody to the A30L protein, or to an epitope appended to its C terminus, to capture complexes from infected cells. A prominent 42-kDa protein was resolved and identified by mass spectrometry as the vaccinia virus G7L protein. This previously uncharacterized protein was expressed late in infection and was associated with immature virions and the cores of mature particles. In order to study the role of the G7L protein, a conditional lethal mutant was made by replacing the G7L gene with an inducible copy. Expression of G7L and formation of infectious virus was dependent on the addition of inducer. Under nonpermissive conditions, morphogenesis was blocked and viral crescent membranes and immature virions containing tubular elements were separated from the electron-dense granular viroplasm, which accumulated in large spherical masses. This phenotype was identical to that previously obtained with an inducible, conditional lethal A30L mutant. Additional in vivo and in vitro experiments provided evidence for the direct interaction of the A30L and G7L proteins and demonstrated that the stability of each one was dependent on its association with the other.

APA, Harvard, Vancouver, ISO, and other styles

42

Li, Jing, Jin Xue, Heng-Mu Zhang, Jian Yang, Li Xie, and Jian-Ping Chen. "Characterization of homologous and heterologous interactions between viroplasm proteins P6 and P9-1 of the fijivirus southern rice black-streaked dwarf virus." Archives of Virology 160, no. 2 (November 7, 2014): 453–57. http://dx.doi.org/10.1007/s00705-014-2268-z.

Full text

APA, Harvard, Vancouver, ISO, and other styles

43

Resch, Wolfgang, Andrea S. Weisberg, and Bernard Moss. "Vaccinia Virus Nonstructural Protein Encoded by the A11R Gene Is Required for Formation of the Virion Membrane." Journal of Virology 79, no. 11 (June 1, 2005): 6598–609. http://dx.doi.org/10.1128/jvi.79.11.6598-6609.2005.

Full text

Abstract:

ABSTRACT The vaccinia virus A11R gene has orthologs in all known poxvirus genomes, and the A11 protein has been previously reported to interact with the putative DNA packaging protein A32 in a yeast two-hybrid screen. Using antisera raised against A11 peptides, we show that the A11 protein was (i) expressed at late times with an apparent mass of 40 kDa, (ii) not incorporated into virus particles, (iii) phosphorylated independently of the viral F10 kinase, (iv) coimmunoprecipitated with A32, and (v) localized to the viral factory. To determine the role of the A11 protein and test whether it is indeed involved in DNA packaging, we constructed a recombinant vaccinia virus with an inducible A11R gene. This recombinant was dependent on inducer for single-cycle growth and plaque formation. In the absence of inducer, viral late proteins were produced at normal levels, but proteolytic processing and other posttranslational modifications of some proteins were inhibited, suggesting a block in virus particle assembly. Consistent with this observation, electron microscopy of cells infected in the absence of inducer showed virus factories with abnormal electron-dense viroplasms and intermediate density regions associated with membranes and containing the D13 protein. However, no viral membrane crescents, immature virions, or mature virions were produced. The requirement for nonvirion protein A11 in order to make normal viral membranes was an unexpected and exciting finding, since neither the origin of these membranes nor their mechanism of formation in the cytoplasm of infected cells is understood.

APA, Harvard, Vancouver, ISO, and other styles

44

Blackhall, J., M. Muñoz, A. Fuentes, and G. Magnusson. "Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation." Journal of Virology 72, no. 8 (August 1, 1998): 6398–405. http://dx.doi.org/10.1128/jvi.72.8.6398-6405.1998.

Full text

Abstract:

ABSTRACT The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.

APA, Harvard, Vancouver, ISO, and other styles

45

Li, Jintao, Haiyang He, and Yuzhang Wu. "Cyclophilin A restricts rotavirus infection by enhancing type 1 interferon response in infected epithelial cells (68.1)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 68.1. http://dx.doi.org/10.4049/jimmunol.188.supp.68.1.

Full text

Abstract:

Abstract Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host protein synthesis, however, previous studies have revealed that RV infection can also lead to the upregulation of host proteins, which could play crucial roles in RV infection. Using proteomic methods, we found that Cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase (PPIase), was upregulated in MA104 cells infected by human rotavirus (HRV). Following infection, CYPA was recruited to the viroplasm where it interacted with HRV structural protein VP2, to inhibit viral protein expressions, thus producing a repressive effect on HRV replication, which was dependent on its PPIase activity. We further show that CYPA was required for host type-1 interferon (IFN-I) response to HRV infection, and this effect was independent on its PPIase activity but dependent on JNK signaling pathway. Furthermore, we found that the expression of CYPA in enterocytes correlates to the period when BALB/c mice become resistannt to RV infection-induced diarrhea. Together, our data identify CYPA as a new host restrictive factor that confers protection against rotavirus infection.

APA, Harvard, Vancouver, ISO, and other styles

46

Szajner, Patricia, Howard Jaffe, Andrea S. Weisberg, and Bernard Moss. "A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions." Virology 330, no. 2 (December 2004): 447–59. http://dx.doi.org/10.1016/j.virol.2004.10.008.

Full text

APA, Harvard, Vancouver, ISO, and other styles

47

Rojas, Margarito, Carlos F. Arias, and Susana López. "Protein Kinase R Is Responsible for the Phosphorylation of eIF2α in Rotavirus Infection." Journal of Virology 84, no. 20 (July 14, 2010): 10457–66. http://dx.doi.org/10.1128/jvi.00625-10.

Full text

Abstract:

ABSTRACT The eukaryotic initiation translation factor 2 (eIF2) represents a key point in the regulation of protein synthesis. This factor delivers the initiator Met-tRNA to the ribosome, a process that is conserved in all eukaryotic cells. Many types of stress reduce global translation by triggering the phosphorylation of the α subunit of eIF2, which reduces the formation of the preinitiation translation complexes. Early during rotavirus infection, eIF2α becomes phosphorylated, and even under these conditions viral protein synthesis is not affected, while most of the cell protein synthesis is blocked. Here, we found that the kinase responsible for the phosphorylation of eIF2α in rotavirus-infected cells is PKR, since in mouse embryonic fibroblasts deficient in the kinase domain of PKR, or in MA104 cells where the expression of PKR was knocked down by RNA interference, eIF2α was not phosphorylated upon rotavirus infection. The viral component responsible for the activation of PKR seems to be viral double-stranded RNA, which is found in the cytoplasm of infected cells, outside viroplasms. Taken together, these results suggest that rotaviruses induce the PKR branch of the interferon system and have evolved a mechanism to translate its proteins, surpassing the block imposed by eIF2α phosphorylation.

APA, Harvard, Vancouver, ISO, and other styles

48

Nejmeddine, M., G. Trugnan, C. Sapin, E. Kohli, L. Svensson, S. Lopez, and J. Cohen. "Rotavirus Spike Protein VP4 Is Present at the Plasma Membrane and Is Associated with Microtubules in Infected Cells." Journal of Virology 74, no. 7 (April 1, 2000): 3313–20. http://dx.doi.org/10.1128/jvi.74.7.3313-3320.2000.

Full text

Abstract:

ABSTRACT VP4 is an unglycosylated protein of the outer layer of the capsid of rotavirus. It forms spikes that project from the outer layer of mature virions, which is mainly constituted by glycoprotein VP7. VP4 has been implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. Previous studies indicated that VP4 is located in the space between the periphery of the viroplasm and the outside of the endoplasmic reticulum in rotavirus-infected cells. Confocal microscopy of infected MA104 monolayers, immunostained with specific monoclonal antibodies, revealed that a significant fraction of VP4 was present at the plasma membrane early after infection. Another fraction of VP4 is cytoplasmic and colocalizes with β-tubulin. Flow cytometry analysis confirmed that at the early stage of viral infection, VP4 was present on the plasma membrane and that its N-terminal region, the VP8* subunit, was accessible to antibodies. Biotin labeling of the infected cell surface monolayer with a cell-impermeable reagent allowed the identification of the noncleaved form of VP4 that was associated with the glycoprotein VP7. The localization of VP4 was not modified in cells transfected with a plasmid allowing the expression of a fusion protein consisting of VP4 and the green fluorescent protein. The present data suggest that VP4 reaches the plasma membrane through the microtubule network and that other viral proteins are dispensable for its targeting and transport.

APA, Harvard, Vancouver, ISO, and other styles

49

Aboughanem-Sabanadzovic, Nina, Thomas W. Allen, James Frelichowski, Jodi Scheffler, and Sead Sabanadzovic. "Discovery and Analyses of Caulimovirid-like Sequences in Upland Cotton (Gossypium hirsutum)." Viruses 15, no. 8 (July 28, 2023): 1643. http://dx.doi.org/10.3390/v15081643.

Full text

Abstract:

Analyses of Illumina-based high-throughput sequencing data generated during characterization of the cotton leafroll dwarf virus population in Mississippi (2020–2022) consistently yielded contigs varying in size (most frequently from 4 to 7 kb) with identical nucleotide content and sharing similarities with reverse transcriptases (RTases) encoded by extant plant pararetroviruses (family Caulimoviridiae). Initial data prompted an in-depth study involving molecular and bioinformatic approaches to characterize the nature and origins of these caulimovirid-like sequences. As a result, here, we report on endogenous viral elements (EVEs) related to extant members of the family Caulimoviridae, integrated into a genome of upland cotton (Gossypium hirsutum), for which we propose the provisional name “endogenous cotton pararetroviral elements” (eCPRVE). Our investigations pinpointed a ~15 kbp-long locus on the A04 chromosome consisting of head-to-head orientated tandem copies located on positive- and negative-sense DNA strands (eCPRVE+ and eCPRVE-). Sequences of the eCPRVE+ comprised nearly complete and slightly decayed genome information, including ORFs coding for the viral movement protein (MP), coat protein (CP), RTase, and transactivator/viroplasm protein (TA). Phylogenetic analyses of major viral proteins suggest that the eCPRVE+ may have been initially derived from a genome of a cognate virus belonging to a putative new genus within the family. Unexpectedly, an identical 15 kb-long locus composed of two eCPRVE copies was also detected in a newly recognized species G. ekmanianum, shedding some light on the relatively recent evolution within the cotton family.

APA, Harvard, Vancouver, ISO, and other styles

50

Chiu, Wen-Ling, and Wen Chang. "Vaccinia Virus J1R Protein: a Viral Membrane Protein That Is Essential for Virion Morphogenesis." Journal of Virology 76, no. 19 (October 1, 2002): 9575–87. http://dx.doi.org/10.1128/jvi.76.19.9575-9587.2002.

Full text

Abstract:

ABSTRACT Vaccinia virus, a member of the poxvirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature virus particles. This study examines the biological function of J1R protein in the vaccinia virus life cycle. A recombinant vaccinia virus was constructed to conditionally express J1R protein in an isopropyl-β-d-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during vaccinia virus infection, the virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that vaccinia virus J1R is a membrane protein that is required for virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'Viroplasmic Proteins'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles