Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

Kiermayer, Simone; Kraus, Ina; Richt, Jürgen A.; Garten, Wolfgang; Eickmann, Markus

doi:10.1016/s0014-5793(02)03513-5

Cited by 17 publications

(19 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As shown in Fig. 1, the molecular mass of GP1 was consistent with that of the wild-type GP1, as described in previous reports [8,12], and all constructs were efficiently expressed in 293T cells.To verify that GP1 binds to BDV-permissive Vero cells but not to non-permissive Chinese hamster ovary (CHO) cells [2], cells were first incubated with 1.5 µg/ml of FLAGtagged GP1, and the binding of this to cells was then detected using an anti-FLAG M2 antibody. Flow cytometric analysis indicated that GP1 bound to Vero but not to CHO cells (Fig.…”

supporting

confidence: 86%

See 1 more Smart Citation

Binding Properties of GP1 Protein of Borna Disease Virus

Makino

Horimoto

Kawaoka

2009

The Journal of Veterinary Medical Science

View full text Add to dashboard Cite

ABSTRACT. The surface glycoprotein (G) of Borna disease virus (BDV) plays central roles in the process of viral entry. BDV G is cleaved by cellular furin-like proteases into two components, GP1 and GP2. Although GP1 is involved in the virus entry into cells, the binding activity of GP1 to cells is unknown. Therefore, we expressed the wild-type GP1 and a variety of GP1 deletion mutants that were FLAGtagged at the C-terminus in human embryonic kidney 293T cells. These proteins were then purified using an anti-FLAG antibody and evaluated for their ability to bind to cell lines. GP1 bound to BDV-permissive cells but not to non-permissive cells. GP1 also inhibited BDV infection via its binding to cells. Borna disease virus (BDV) causes a disease of the central nervous system that is found primarily in horses and sheep and is characterized by behavioral abnormalities and diverse pathology [10,15]. BDV is the only member of Bornaviridae, which is an enveloped virus with a negative sense nonsegmented RNA genome of approximately 8.9 kb [9].BDV entry into the host cells is mediated by the viral glycoprotein (G) via receptor-dependent endocytosis [1,5,6]. The BDV G gene is initially synthesized as a 56 to 57 kDa polypeptide precursor and is modified by N-glycosylation in the endoplasmic reticulum and Golgi apparatus, resulting in a protein with a molecular weight of up to 95 kDa [4,14]. Cellular furin-like proteases cleave the G protein into the ectodomain subunit GP1 and the transmembrane subunit GP2 [13]. GP1 (51-60 kDa) is highly glycosylated and sufficient to mediate viral entry [8,11]. GP2 (43 kDa) has a putative fusion domain at the N-terminus that is involved in the pH-dependent fusion event [6].BDV GP1 fused to the transmembrane and cytoplasmic domain of vesicular stomatitis virus glycoprotein (VSV G) can mediate the cellular entry of VSV∆G, which is a mutant VSV lacking the G gene [11]. However, the binding activity of GP1 to cells remains unknown. Therefore, we attempted to evaluate the binding of the BDV GP1 subunit to cells by generating and analyzing a series of GP1 truncation mutants.BDV GP1 (amino acid residues 1 to 244) and several GP1 truncation mutants were generated via polymerase chain reaction using the BDV G target from the laboratory strain He/80 [3] and cloned into an expression vector (pCAGGS) with a FLAG tag at the C-terminus of the polypeptide. The extracellular domain of GP2 (amino acid residues 250 to 468) with signal peptide at the N-terminus and a FLAG tag at the C-terminus was also inserted into pCAGGS. The GP1 truncation mutants used in this study are presented in Fig. 1A.In order to purify the various BDV G fragments, human embryonic kidney 293T cells were transfected with the plasmids encoding FLAG-tagged GP1 or GP2 fragments by the TransIT-293 reagent (Mirus Bio Corp, Billerica, MA, U.S.A.). At 24 hr post-transfection, the cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and the protease inhibitor cocktail Complete Mini [Roche, Basel,...

show abstract

supporting

confidence: 86%

“…Cellular furin-like proteases cleave the G protein into the ectodomain subunit GP1 and the transmembrane subunit GP2 [13]. GP1 (51-60 kDa) is highly glycosylated and sufficient to mediate viral entry [8,11]. GP2 (43 kDa) has a putative fusion domain at the N-terminus that is involved in the pH-dependent fusion event [6].…”

mentioning

confidence: 99%

Binding Properties of GP1 Protein of Borna Disease Virus

Makino

Horimoto

Kawaoka

2009

The Journal of Veterinary Medical Science

View full text Add to dashboard Cite

show abstract

“…7B). This protein likely corresponds to a partially glycosylated GPC, suggesting that furin-mediated maturation of BDV G may be involved in the complete glycosylation of BDV G. We have generated a novel Ab (Ab-GP1 N ) capable of detecting GP1 efficiently without the need of endoglycosidase treatments (16), which allowed us to examine the levels of GP1 in BDVinfected cells and virions. The use of Ab-GP1 N , together with the previously described Ab-G (11), revealed that all three BDV G species, GPC, GP1, and GP2, are incorporated into virions.…”

Section: Discussionmentioning

confidence: 99%

“…GPC is posttranslationally cleaved by the cellular protease furin into GP1 (GP N ) and GP2 (GP C ), which correspond to the N-and C-terminal regions, respectively, of GPC (9,27). GPC and GP2 are readily detected in both infected cells and cell-free virions (9,10), whereas antibody (Ab) detection of GP1 has been complicated by its high content of N-glycans that shield antigenic sites (16).…”

mentioning

confidence: 99%

Cell-to-Cell Spread of Borna Disease Virus Proceeds in the Absence of the Virus Primary Receptor and Furin-Mediated Processing of the Virus Surface Glycoprotein

Clemente

Torre

2007

J Virol

View full text Add to dashboard Cite

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of Mononegavirales. BDV cell entry follows a receptor-mediated endocytosis pathway, which is initiated by the recognition of an as-yet-unidentified receptor at the cell surface by the virus glycoprotein G. BDV G is synthesized as a precursor (GPC) that is cleaved by the cellular protease furin to produce the mature glycoproteins GP1 and GP2, which have been implicated in receptor recognition and pH-dependent fusion events, respectively. BDV is highly neurotropic and its spread in cultured cells proceeds in the absence of detectable extracellular virus or syncytium formation. BDV spread has been proposed to be strictly dependent on the expression and correct processing of BDV G. Here we present evidence that cell-to-cell spread of BDV required neither the expression of cellular receptors involved in virus primary infection, nor the furin-mediated processing of BDV G. We also show that in furin-deficient cells, the release of BDV particles induced by the treatment of BDV-infected cells with hypertonic buffer was not significantly affected, while virion infectivity was dramatically impaired, correlating with the decreased incorporation of BDV G species into viral particles. These findings support the view that the propagation of BDV within the central nervous systems of infected hosts involves both a primary infection that follows a receptor-mediated endocytosis pathway and a subsequent cell-to-cell spread that is independent of the expression of the primary receptor and does not require the processing of BDV G into GP1 and GP2.

show abstract

“…The C-terminus product (GP2) is readily detected in virally infected cells and migrates with an M r of 43,000. In contrast, detection of the predicted N-terminal product (GP1) of G has been difficult due to its high content of N glycans, which shield antigenic sites recognized by antibodies (18). GPC accumulates in the endoplasmic reticulum and perinuclear region, but it is typically not detected on the plasma membrane, whereas GP2 accumulates at the cell surface (13).…”

mentioning

confidence: 99%

Generation and Characterization of a Recombinant Vesicular Stomatitis Virus Expressing the Glycoprotein of Borna Disease Virus

Perez

Clemente

Jeetendra

et al. 2007

J Virol

View full text Add to dashboard Cite

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSV⌬G*/BDVG). Cells infected with rVSV⌬G*/BDVG produced high titers (10 7 PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSV⌬G*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSV⌬G*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSV⌬G*/BDVG-infected cells. Notably, rVSV⌬G*/ BDVG recreated the cell tropism and entry pathway of bona fide BDV. Our results indicate that rVSV⌬G*/ BDVG represents a unique tool for the investigation of BDV G-mediated cell entry, as well as the roles of BDV G in host immune responses and pathogenesis associated with BDV infection.Borna disease virus (BDV) causes central nervous system (CNS) disease in a variety of vertebrate species, frequently manifested by behavioral abnormalities (31). BDV was originally identified as the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep within regions of endemicity in central Europe (31). Current epidemiological data, however, indicate that the natural host range, prevalence, and geographic distribution of BDV are broader than originally thought (8,16,20,29). Experimentally, BDV has a wide host range, from birds to rodents and nonhuman primates (8,10,16,20,29). Both host and viral factors contribute to a variable period of incubation and significant heterogeneity in the symptoms and pathology associated with infection (8,10,16,20,29).BDV is an enveloped virus with a nonsegmented negativestrand RNA genome with a characteristic mononegavirales organization (4, 34, 35). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. The BDV genome (ca 8.9 kb) contains six major open reading frames (ORFs) in the order 3Ј-N-p10/P-M-G-L-5Ј (4, 34, 35). These ORFs code for the virus nucleoprotein (N), phosphoprotein (P) transcriptional activator, matrix (M), surface glyc...

show abstract

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

Cited by 17 publications

References 15 publications

Binding Properties of GP1 Protein of Borna Disease Virus

Binding Properties of GP1 Protein of Borna Disease Virus

Cell-to-Cell Spread of Borna Disease Virus Proceeds in the Absence of the Virus Primary Receptor and Furin-Mediated Processing of the Virus Surface Glycoprotein

Generation and Characterization of a Recombinant Vesicular Stomatitis Virus Expressing the Glycoprotein of Borna Disease Virus

Contact Info

Product

Resources

About