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  • 1
    Online Resource
    Online Resource
    Lack of Processing of the Expressed ORF1 Gene Product of Hepatitis E Virus
    Springer Science and Business Media LLC ; 2011
    In:  Virology Journal Vol. 8, No. 1 ( 2011-12)
    Details
    In: Virology Journal, Springer Science and Business Media LLC, Vol. 8, No. 1 ( 2011-12)
    Abstract: Proteolytic processing is a common mechanism among plus strand RNA viruses and the replicases of all plus strand RNA viruses of animals thus far characterized undergo such processing. The replicase proteins of hepatitis E virus (HEV) are encoded by ORF1. A previous report published by our group [1] provided data that processing potentially occurred when ORF1 (Burma strain; genotype 1) was expressed using a vaccinia virus-based expression system. Findings To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed. When ORF1 from an HEV infectious cDNA clone (US swine strain; genotype 3) was expressed using the plasmid-based system, the only species detected was the 185 kDa precursor of ORF1. A putative papain-like cysteine protease [2] had been predicted within ORF1 using the original HEV genomic sequence. However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved. Conclusions The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.
    Type of Medium: Online Resource
    ISSN: 1743-422X
    URL: Article
    DOI: 10.1186/1743-422X-8-245
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2011
    detail.hit.zdb_id: 2160640-7
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  • 2
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    Analysis of Rubella Virus Capsid Protein-Mediated Enhancement of Replicon Replication and Mutant Rescue
    American Society for Microbiology ; 2006
    In:  Journal of Virology Vol. 80, No. 8 ( 2006-04-15), p. 3966-3974
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 80, No. 8 ( 2006-04-15), p. 3966-3974
    Abstract: The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed ΔNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (CΔ8-Vero cells), a construct previously shown to be unable to complement ΔNotI. In C-Vero cells but not Vero or CΔ8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with ΔNotI (RUBrep/GFP-ΔNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5′ and 3′ cis -acting elements were also rescued in C-Vero cells. Interestingly, the CΔ8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in CΔ8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.80.8.3966-3974.2006
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
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  • 3
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    The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans
    American Society for Microbiology ; 1998
    In:  Journal of Virology Vol. 72, No. 5 ( 1998-05), p. 4463-4466
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 72, No. 5 ( 1998-05), p. 4463-4466
    Abstract: The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn 2+ , Cd 2+ , and Co 2+ , the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.72.5.4463-4466.1998
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
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  • 4
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    Online Resource
    Mutagenic Analysis of the 3′ cis -Acting Elements of the Rubella Virus Genome
    American Society for Microbiology ; 1999
    In:  Journal of Virology Vol. 73, No. 4 ( 1999-04), p. 3386-3403
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 73, No. 4 ( 1999-04), p. 3386-3403
    Abstract: Thermodynamically predicted secondary structure analysis of the 3′-terminal 305 nucleotides (nt) of the rubella virus (RUB) genome, a region conserved in all RUB defective interfering RNAs, revealed four stem-loop (SL) structures; SL1 and SL2 are both located in the E1 coding region, while SL3 and SL4 are within the 59-nt 3′ untranslated region (UTR) preceding the poly(A) tract. SL2 is a structure shown to interact with human calreticulin (CAL), an autoantigen potentially involved in RUB RNA replication and pathogenesis. RNase mapping indicated that SL2 and SL3 are in equilibrium between two conformations, in the second of which the previously proposed CAL binding site in SL2, a U-U bulge, is not formed. Site-directed mutagenesis of the 3′ UTR with a RUB infectious clone, Robo302, revealed that most of the 3′ UTR is required for viral viability except for the 3′-terminal 5 nt and the poly(A) tract, although poly(A) was rapidly regenerated during subsequent replication. Maintenance of the overall SL3 structure, the 11-nt single-stranded sequence between SL3 and SL4, and the sequences forming SL4 were all important for viral viability. Studies on the interaction between host factors and the 3′ UTR showed the formation of three RNA-protein complexes by gel mobility shift assay, and UV-induced cross-linking detected six host protein species, with molecular masses of 120, 80, 66, 55, 48, and 36 kDa, interacting with the 3′ UTR. Site-directed mutagenesis of SL2 by nucleotide substitutions showed that maintenance of SL2 stem rather than the U-U bulge was critical in CAL binding since mutants having the U-U bulge base paired had a similar binding activity for CAL as the native structure whereas mutants having the SL2 stem destabilized had much lower binding activity. However, all of these mutations gave rise to viable viruses when introduced into Robo302, indicating that binding of CAL to SL2 is independent of viral viability.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.73.4.3386-3403.1999
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1999
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  • 5
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    Online Resource
    Time course of virus-specific macromolecular synthesis during rubella virus infection in vero cells
    Elsevier BV ; 1988
    In:  Virology Vol. 162, No. 1 ( 1988-1), p. 65-75
    Details
    In: Virology, Elsevier BV, Vol. 162, No. 1 ( 1988-1), p. 65-75
    Type of Medium: Online Resource
    ISSN: 0042-6822
    URL: Article
    DOI: 10.1016/0042-6822(88)90395-9
    RVK:
    WA 15000
    Language: English
    Publisher: Elsevier BV
    Publication Date: 1988
    detail.hit.zdb_id: 1471925-3
    SSG: 12
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  • 6
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    Online Resource
    Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells
    Springer Science and Business Media LLC ; 2014
    In:  BMC Research Notes Vol. 7, No. 1 ( 2014-12)
    Details
    In: BMC Research Notes, Springer Science and Business Media LLC, Vol. 7, No. 1 ( 2014-12)
    Type of Medium: Online Resource
    ISSN: 1756-0500
    URL: Article
    DOI: 10.1186/1756-0500-7-757
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2014
    detail.hit.zdb_id: 2413336-X
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  • 7
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    Cryo-Electron Tomography of Rubella Virus
    American Society for Microbiology ; 2012
    In:  Journal of Virology Vol. 86, No. 20 ( 2012-10-15), p. 11078-11085
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 86, No. 20 ( 2012-10-15), p. 11078-11085
    Abstract: Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus . The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.01390-12
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
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  • 8
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    Online Resource
    Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease
    American Society for Microbiology ; 2000
    In:  Journal of Virology Vol. 74, No. 13 ( 2000-07), p. 5949-5956
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 74, No. 13 ( 2000-07), p. 5949-5956
    Abstract: The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463–4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn 65 binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV–CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.74.13.5949-5956.2000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2000
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  • 9
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    Analyses of Phosphorylation Events in the Rubella Virus Capsid Protein: Role in Early Replication Events
    American Society for Microbiology ; 2006
    In:  Journal of Virology Vol. 80, No. 14 ( 2006-07-15), p. 6917-6925
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 80, No. 14 ( 2006-07-15), p. 6917-6925
    Abstract: The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.01152-05
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
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  • 10
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    A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication
    Portland Press Ltd. ; 2009
    In:  Biochemical Journal Vol. 417, No. 2 ( 2009-01-15), p. 477-483
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 417, No. 2 ( 2009-01-15), p. 477-483
    Abstract: The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn2+-binding ligands. This minidomain bound to Zn2+ with a stoichiometry of ≈0.7 and an apparent dissociation constant of & lt;500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Zn2+ binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cys1175, Cys1178, Cys1225 and Cys1227 were required for the binding of Zn2+. Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)48CXC Zn2+-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20081468
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2009
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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