A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A and alphaviruses fails to cleave Sindbis virus glycoprotein PE2

Watson, David; Moehring, Joan M.; Moehring, Thomas J.

doi:10.1128/jvi.65.5.2332-2339.1991

Cited by 47 publications

(35 citation statements)

References 33 publications

(31 reference statements)

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“…In other viruses the fusion proteins are initially held in an inactive state by their association with a second protein, and the activation step requires the proteolytic cleavage of this second protein and not of the fusion protein itself. Such a mechanism has been demonstrated for alphaviruses (Lobigs and Garoff, 1990;Watson et al, 1991;Salminen et al, 1992), and there is now evidence that flaviviruses use a similar mechanism based on the interaction between prM and E to switch from a fusion-inactive form during assembly to the fusion-competent form required for entry.…”

Section: J Function Of Prm Proteinmentioning

confidence: 88%

Structures and mechanisms in flavivirus fusion

Heinz

Allison

2000

Advances in Virus Research

150

106

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Section: J Function Of Prm Proteinmentioning

confidence: 88%

Structures and mechanisms in flavivirus fusion

Heinz

Allison

2000

Advances in Virus Research

150

106

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“…Besides its role in the maturation of cellular proteins, furin can cleave a variety of viral structural proteins and several bacterial toxins (Hallenberger et al, 1992;Stieneke-Gröber et al, 1992;Klimpel et al, 1992;Gotoh et al, 1992;Tsuneoka et al, 1993;Gordon and Leppla, 1994). Furin-deficient cells are often resistant to viral infection and not killed by toxins (Watson et al, 1991). The biological relevance of furin activity has been established by showing that resistance to toxins and viruses can be overcome by the transfection of a furin gene (Moehring et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine‐rich loop of diphtheria toxin

Chiron

Ogata

FitzGerald

1996

Molecular Microbiology

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To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro, furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5-10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0-5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.

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“…Spike assembly occurs after E1␤ formation and is required for E1 acquisition of resistance to the membrane-permeative reducing agent dithiothreitol (DTT) (8)(9)(10)34) and export of E1 from the ER (8,10,23,31). Prior to the arrival of the spike at the cell surface, PE2 is processed to E2 by a furin-like proteinase resident within the trans-Golgi network (3,14,46). Therefore, the cleavage of E3 from PE2 can be used to assay the export of the spike from the ER in ts23-infected cells (8).…”

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confidence: 99%

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype

Carleton

Brown

1997

J Virol

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The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.

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A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A and alphaviruses fails to cleave Sindbis virus glycoprotein PE2

Cited by 47 publications

References 33 publications

Structures and mechanisms in flavivirus fusion

Structures and mechanisms in flavivirus fusion

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine‐rich loop of diphtheria toxin

The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype

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