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

2018 ◽  
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.

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

2018 ◽  
Vol 92 (23) ◽  
Author(s):  
Altar M. Munis ◽  
Maha Tijani ◽  
Mark Hassall ◽  
Giada Mattiuzzo ◽  
Mary K. Collins ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Envelope Glycoprotein ◽  
Lentiviral Vectors ◽  
Medicinal Products ◽  
Vaccine Vectors ◽  
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 over VSVind.G. Well-characterized antibodies that recognize 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 neutralizes 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 neutralizes 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 neutralization. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its postfusion structure, and we propose that 8G5F11 cross-neutralizes VesGs by inhibiting this.IMPORTANCEVSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.

scholarly journals Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein

2009 ◽  
Vol 83 (23) ◽  
pp. 12139-12150 ◽  
Author(s):  
Chad E. Mire ◽  
Derek Dube ◽  
Sue E. Delos ◽  
Judith M. White ◽  
Michael A. Whitt
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Conformational Changes ◽  
Matrix Protein ◽  
Low Ph ◽  
M Protein ◽  
Steady Increase ◽  
Protein Dissociation ◽  
Vesicular Stomatitis

ABSTRACT To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since “bald” ΔG-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating.

scholarly journals Posttranslational folding of vesicular stomatitis virus G protein in the ER: involvement of noncovalent and covalent complexes.

1993 ◽  
Vol 120 (3) ◽  
pp. 647-655 ◽  
Author(s):  
A de Silva ◽  
I Braakman ◽  
A Helenius
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Chain Termination ◽  
Metabolic Energy ◽  
Folding Pathway ◽  
Protein Subunits ◽  
Late Step ◽  
Vesicular Stomatitis ◽  
Transient Intermediates

In this study, we show that posttranslational folding of Vesicular Stomatitis virus G protein subunits can involve noncovalent, multimeric complexes as transient intermediates. The complexes are heterogeneous in size (4-21S20,W), contain several G glycopolypeptides, and are associated with BiP/GRP78. The newly synthesized, partially intrachain disulfide-bonded G proteins enter these complexes immediately after chain termination, and are released 1-4 min later as fully oxidized, trimerization-competent monomers. These monomers are properly folded, judging by their binding of conformation-specific mAbs. When the G protein is translated in the presence of DTT, it remains reduced, largely unfolded and aggregated in the ER, but it can fold successfully when the DTT is removed. In this case, contrary to normal folding, the aggregates become transiently disulfide cross-linked. We also demonstrated that the fidelity of the folding process is dependent on metabolic energy. Finally, we established that the G protein of the folding mutant of the Vesicular Stomatitis virus, ts045, is blocked at a relatively late step in the folding pathway and remains associated with oligomeric, BiP/GRP78-containing folding complexes.

scholarly journals Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

1989 ◽  
Vol 108 (3) ◽  
pp. 811-819 ◽  
Author(s):  
K Suh ◽  
J E Bergmann ◽  
C A Gabel
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Plasmid Vector ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
High Mannose

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

scholarly journals Efficient Export of the Vesicular Stomatitis Virus G Protein from the Endoplasmic Reticulum Requires a Signal in the Cytoplasmic Tail That Includes Both Tyrosine-based and Di-acidic Motifs

2000 ◽  
Vol 11 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Carolyn S. Sevier ◽  
Ora A. Weisz ◽  
Mollie Davis ◽  
Carolyn E. Machamer
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Cytoplasmic Tail ◽  
Reporter Protein ◽  
Transmembrane Glycoprotein ◽  
Vesicular Stomatitis ◽  
Er Export

The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxØ (where x is any amino acid and Ø is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.

Sign in / Sign up

Export Citation Format

Share Document