Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis

Geiselhart, Verena; Schwantes, Astrid; Bastone, Patrizia; Frech, Matthias; Löchelt, Martin

doi:10.1016/s0042-6822(03)00125-9

Cited by 40 publications

(48 citation statements)

References 25 publications

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“…As reported previously (11) and in agreement with the data presented here, FV particles contain high concentrations of the 16-kDa Elp but low levels of the 9-kDa FFV Env SP. The N-terminal sequences extending into the cytosol before budding and thereafter into the virion are identical for both proteins.…”

Section: Discussionsupporting

confidence: 93%

“…However, for two distinct foamy retroviruses, namely human and feline foamy virus (HFV and FFV), the 14-to 18-kDa N-terminal Env cleavage product was clearly detectable as a component of released virus particles (11,14,15,35). We showed that the N terminus of FFV Env, termed the Env leader protein (Elp), is located inside particles, confirming that specific interactions between this part of Elp and Gag can occur (11,35).…”

mentioning

confidence: 75%

“…For preparative purification of FFV Elp, supernatants from 25 150-cm 2 flasks of FFV-infected CRFK cells harvested 5 days postinfection were used to enrich FFV particles by centrifugation through sucrose and banding in Optiprep as described previously (11). The pelleted virions were denatured, deglycosylated in 100 l with 1 U of PNGase F (see below), and separated on precast 12% NuPAGE gels (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…We showed that the N terminus of FFV Env, termed the Env leader protein (Elp), is located inside particles, confirming that specific interactions between this part of Elp and Gag can occur (11,35). Thus, the unusually large size and intrinsic stability of the N-terminal cleavage product of foamy virus (FV) Env indicate that it is significantly different from the classical SPs of cellular or viral proteins.…”

mentioning

confidence: 94%

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Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

Geiselhart¹,

Bastone²,

Kempf

et al. 2004

J Virol

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The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.In retroviruses, proteolytic processing of the structural Gag and enzymatic Pol proteins is executed by the virus-encoded protease (6). In contrast, the Env glycoprotein precursors are considered to be processed by two cellular proteases (8,13,19). The first cleavage occurring in the endoplasmic reticulum (ER) removes the signal peptide (SP). In analogy to cellular secretory or transmembrane (TM) proteins, proteolytic removal of the SP which targets the Env precursor to the secretory pathway via the ER and Golgi apparatus is considered to be performed by cellular signal peptidases (SPases) (for a review, see references 17 and 19). The second cleavage, which separates the surface (SU) and TM domains, is catalyzed by a member of the furin convertase protease family, often the furin protease itself (8,28). Related glycoprotein processing mechanisms also apply to a wide variety of other membrane viruses such as influenza virus and Ebola virus (4).The processing of cellular type I membrane proteins (or even polyprotein precursors) by ER-resident SPases has been exploited by many viruses leading to SPs of about 15 to more than 50 amino acid residues independent of whether the protein is of viral or cellular origin (17). In addition, the SP is rapidly degraded by signal peptide peptidases, a process which is often directly linked to the presentation of the corresponding cleavage products by the major histocompatibility complex I for purposes of immune surveillance (17).However, for two distinct foamy retroviruses, namely human and feline foamy virus (HFV and FFV), the 14-to 18-kDa N-terminal Env cleavage product was clearly detectable as a component of released virus particles (11,14,15,35). We showed that the N terminus of FFV Env, termed the Env leader protein (Elp), is located inside particles, confirming that specific interact...

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Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 94%

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Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

Geiselhart¹,

Bastone²,

Kempf

et al. 2004

J Virol

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“…The envelope (Env) proteins of the foamy viruses differ in numerous points from those of other retroviruses. They consist of the Env leader protein (Elp) [12], the surface envelope protein and the TM protein [13]. The TM proteins are larger, their central part is characterised by the presence of seven to eight cysteines and numerous potential glycosylation sites [14,15] and electron microscopic pictures demonstrate an unusual trimeric oligomerisation [16].…”

Section: Introductionmentioning

confidence: 99%

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

Mühle

Löchelt

Denner

2012

Protein Expression and Purification

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The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy virus using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocols, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (Muehle et al., Virology, 412:333-340, 2011), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.

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Foamy Viruses

Rethwilm

2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis

Cited by 40 publications

References 25 publications

Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

Foamy Viruses

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