Expression of the Rubella Virus Nonstructural Protein ORF and Demonstration of Proteolytic Processing

Marr, Lee D.; Wang, Chin-Yen; Frey, Teryl K.

doi:10.1006/viro.1994.1070

Cited by 46 publications

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“…Plasmids pTM1/nsRUB and pTM1/nsRUB*, in which transcription and translation of the RUB NSP ORF are under control of the T7 polymerase promoter and the internal ribosome entry site of encephalomyocarditis virus, respectively, as well as Robo302, a RUB infectious cDNA clone, were described previously (18,19,20). pTM1/ nsRUB* contains a Cys1152Gly mutation which abrogates NS protease activity.…”

Section: Methodsmentioning

confidence: 99%

“…Figure 4A and Table 4 show that the Cys1167Ser, Cys1175Ser, Cys1178Ser, His1204Leu, and Cys1227Ser mutations resulted in complete abolition of cleavage. In addition, it has previously been shown that Cys1152 and His1273 are also essential amino acids for protease function (5,19). Mutation of four of these, Cys1175, Cys1178, Cys1227, and His1273, inhibited zinc binding.…”

Section: Fig 2 Activity Of Ns Protease Expressed As a Bacterial Fusmentioning

confidence: 99%

“…A limited homology exists between a domain just upstream from the cleavage site and the active site of papain-like cysteine proteases (PCPs) (9). Within this domain, Cys1152 and His1273 were predicted to form the catalytic dyad and site-directed mutagenesis confirmed that both residues are essential for protease activity (5,19). While activity was readily detected when the protease domain was expressed in vivo, activity was never observed in vitro.…”

mentioning

confidence: 99%

“…Computer-assisted comparison of the RUB NSP ORF with other plus-strand RNA viruses revealed a number of shared consensus motifs, namely, methyltransferase, protease, helicase, and replicase domains. Of these, only the protease and helicase domains have been characterized biochemically (5,10,19).…”

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confidence: 99%

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Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Liu¹,

Yang²,

Ghazi

et al. 2000

J Virol

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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 (

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Section: Methodsmentioning

confidence: 99%

Section: Fig 2 Activity Of Ns Protease Expressed As a Bacterial Fusmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Liu¹,

Yang²,

Ghazi

et al. 2000

J Virol

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“…While in many cases the specific roles of capsid phosphorylation have yet to be established, this posttranslational modification is known to affect genome packaging (9,13,16,17,20) and subcellular localization of capsid proteins (18,19,25,30). Interestingly, it was first reported more than 30 years ago that togavirus capsid proteins are phosphorylated (27,37). However, surprisingly little is known about which amino acid residues in the capsids are modified and how phosphorylation regulates the functions of these proteins.…”

Section: Discussionmentioning

confidence: 99%

Analyses of Phosphorylation Events in the Rubella Virus Capsid Protein: Role in Early Replication Events

Law¹,

Ilkow²,

Tzeng

et al. 2006

J Virol

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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.Rubella virus (RV) is the sole member of the genus Rubivirus within the family Togaviridae. In most cases, RV infection causes a mild self-limiting disease in humans known as rubella or German measles. However, in contrast to postnatal infection, which is relatively benign in nature, in utero infection can have serious consequences for the developing fetus (6). When contracted during the first trimester of pregnancy, RV infection results in a characteristic series of severe defects in the neonate known as congenital rubella syndrome. Despite the availability of an effective vaccine, RV remains a threat to public health in both developed and developing countries as a result of unvaccinated populations. The mechanisms by which RV causes developmental abnormalities are largely unknown.As with all togaviruses, the RV genome contains two open reading frames that encode the replicase proteins and the structural proteins (reviewed in reference 11). The structural proteins are translated as a polyprotein precursor from a subgenomic RNA. The precursor protein is then processed into three structural proteins: a capsid protein and two envelope glycoproteins, E2 and E1. Recently, our laboratory has focused largely on the processing and functional analyses of the capsid protein.During virus assembly, the capsid is thought to interact with viral genomic RNA and glycoproteins E2 and E1 to drive formation of the nucleocapsid and virus budding, respectively. Nucleocapsid formation is highly regulated in RV-infected cells and is coincident with virus budding (29, 32). The packaging signal within the RV genomic RNA and the capsid region that binds this sequence have been identified (24). Moreover, data from our lab suggest that phosphorylation of serine 46 within the RNA binding site of the capsid regulates interactions between the capsid and genomic RNA and, by extrapolation, nucleocapsid formation (22). Mutation of serine 46 to alanine results in capsids that are poorly phosphorylated and ...

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Topley &Amp; Wilson's Microbiology and Microbial Infections

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Expression of the Rubella Virus Nonstructural Protein ORF and Demonstration of Proteolytic Processing

Cited by 46 publications

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Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Analyses of Phosphorylation Events in the Rubella Virus Capsid Protein: Role in Early Replication Events

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