Interactions of a vesicular stomatitis virus G protein fragment with phosphatidylserine: NMR and fluorescence studies

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

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Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2869-2874.1988 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2869-2874

Author(s):

J L Guan ◽

A Ruusala ◽

H Cao ◽

J K Rose

Keyword(s):

Endoplasmic Reticulum ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Cytoplasmic Domain ◽

Secretory Proteins ◽

Vesicular Stomatitis Virus Glycoprotein ◽

Virus Glycoprotein ◽

Vesicular Stomatitis

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.

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Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.

The Journal of Cell Biology ◽

10.1083/jcb.133.3.543 ◽

1996 ◽

Vol 133 (3) ◽

pp. 543-558 ◽

Cited By ~ 119

Author(s):

A Müsch ◽

H Xu ◽

D Shields ◽

E Rodriguez-Boulan

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Mdck Cells ◽

Current Model ◽

Gh3 Cells ◽

Plasma Membrane Proteins ◽

Vesicular Release ◽

Vesicular Stomatitis

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.

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Characterisation of Antibody Interactions with the G Protein of Vesicular Stomatitis Virus Indiana Strain and Other Vesiculovirus G Proteins

10.1101/330910 ◽

2018 ◽

Cited By ~ 1

Author(s):

Altar M Munis ◽

Maha Tijani ◽

Mark Hassall ◽

Giada Mattiuzzo ◽

Mary K Collins ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

Vesicular Stomatitis Virus ◽

Conformational Changes ◽

Lentiviral Vectors ◽

Medicinal Products ◽

Vaccine Vectors ◽

Receptor Binding Site ◽

Advanced Therapy ◽

Vesicular Stomatitis

ABSTRACTVesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example Cocal virus, have been proposed as alternative LV envelopes with possible advantages compared to VSVind.G. Well-characterised antibodies that recognise VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralises G proteins from three strains of VSV as well as Cocal, and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralises only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralisation. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its post-fusion structure, and we propose that 8G5F11 cross-neutralises VesGs by inhibiting this.IMPORTANCEVSVind.G is currently regarded as the gold-standard envelope to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, and identified the epitopes they recognise, and explored the mechanisms behind their neutralisation activity. Understanding how cross-neutralising antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and co-evolution as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.

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Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

The Journal of Cell Biology ◽

10.1083/jcb.102.6.2147 ◽

1986 ◽

Vol 102 (6) ◽

pp. 2147-2157 ◽

Cited By ~ 24

Author(s):

L Puddington ◽

C E Machamer ◽

J K Rose

Keyword(s):

Plasma Membrane ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Heavy Chain ◽

Cytoplasmic Domain ◽

Integral Membrane Proteins ◽

Vesicular Stomatitis Virus Glycoprotein ◽

Hybrid Proteins ◽

Vesicular Stomatitis ◽

Immunoglobulin Mu

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

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