Formation and Assembly of Alphavirus Glycoproteins

Schlesinger, Milton J.; Schlesinger, Sondra

doi:10.1007/978-1-4757-0785-4_5

Cited by 70 publications

(57 citation statements)

References 123 publications

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“…SFV and other alphaviruses such as Sindbis virus and Ross River virus (Cheng et al, 1995;Kielian & Helenius, 1986;Schlesinger & Schlesinger, 1986;Zhang et al, 2002) assemble in a welldefined manner (Kielian, 1995;Kielian & Helenius, 1986;Kielian et al, 1990;Schlesinger & Schlesinger, 1986;Simons & Garoff, 1980;Simons & Warren, 1984). A capsid is formed in the cytoplasm during incorporation of the single strand of genomic RNA (Simons & Warren, 1984).…”

Section: The Role Of Rafts In Virus Assemblymentioning

confidence: 99%

Do lipid rafts mediate virus assembly and pseudotyping?

Briggs

Wilk

Fuller

2003

Journal of General Virology

113

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Section: The Role Of Rafts In Virus Assemblymentioning

confidence: 99%

Do lipid rafts mediate virus assembly and pseudotyping?

Briggs

Wilk

Fuller

2003

Journal of General Virology

113

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“…The SFV system supplies a multitude of advantages for the expression of recombinant proteins in mammalian cells: (i) large-scale production for up to 72 hours post-transfection; (ii) a broad host range; (iii) modifications such as glycosylation in a correct and sufficient way; and (iv) an easy and fast transfection procedure with in-vitro-transcribed RNA. SFV is an insect-borne alphavirus and belongs to the family of Togaviridae (Schlesinger, 1986). Its viral genome consists of capped and polyadenylated single-stranded RNA of positive polarity and encodes its own RNA polymerase.…”

Section: Prion-like Protein Doppel Causes Purkinje Cell Degeneration Inmentioning

confidence: 99%

Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance

Gauczynski¹,

Krasemann²,

Bodemer

et al. 2002

Journal of Cell Science

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The Semliki-Forest virus (SFV) system was used to overexpress human wild-type and mutant prion proteins as well as FLAG-tagged human and bovine PrP in mammalian cells. The application of recombinant SFV vectors allowed a high-level production of highly glycosylated prion proteins with a molecular weight ranging from 25 to 30 kDa for recombinant wild-type human PrP and from 26 to 32 kDa for wild-type bovine PrP. Further, we report here the generation of recombinant mutant prion proteins that are associated with inherited human prion diseases such as fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Both mutated variants, the FFI-associated PrP carrying a mutation at amino acid position 178 and the CJD-linked form containing an insertion of nine additional octarepeats reveal proteinase K resistance, one of the typical biochemical properties of the infectious scrapie isoform of the prion protein. By contrast, recombinant wild-type PrP was completely proteinase K sensitive when expressed in SFV-transfected BHK cells. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP. In order to purify recombinant human and bovine PrP from cell lysates, a FLAG-tag was introduced either at the N-terminus behind the signal peptide or at the C-terminus close to the adhesion site of the GPI anchor. N-terminal insertion did not extensively influence the trafficking of the FLAG-tagged protein to the cell surface, whereas insertion close to the GPI attachment site clearly affected the transport of the majority of PrP to the cell membrane, probably resulting in their retention within the secretory pathway. All FLAG-tagged prion proteins were expressed efficiently in BHK cells and showed a typical glycosylation pattern, allowing their rapid and simple purification via anti-FLAG antibody chromatography.

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“…Fusion is mediated by the viral spike protein which is a heterotrimer of two trans-membrane subunits, E1 and E2, plus a peripheral protein E3 (reviewed in Schlesinger & Schlesinger, 1986;Simons & Warren, 1984). E1 is considered to be the fusion protein since virus particles devoid of E2 and E3 after trypsin treatment are still infectious (Omar & Koblet, 1988) and E2/E3 expressed alone are fusion negative (KondorKoch et al, 1983).…”

Section: Generalmentioning

confidence: 99%