Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus

Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.

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Matrix Protein of Rabies Virus Is Responsible for the Assembly and Budding of Bullet-Shaped Particles and Interacts with the Transmembrane Spike Glycoprotein G

Journal of Virology ◽

10.1128/jvi.73.1.242-250.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 242-250 ◽

Cited By ~ 181

Author(s):

Teshome Mebatsion ◽

Frank Weiland ◽

Karl-Klaus Conzelmann

Keyword(s):

Rabies Virus ◽

Matrix Protein ◽

M Protein ◽

Virus Budding ◽

Glycoprotein G ◽

Severe Impairment ◽

Rnp Complex ◽

Long Rod ◽

Nucleoprotein N

ABSTRACT To elucidate the functions of rhabdovirus matrix (M) protein, we determined the localization of M in rabies virus (RV) and analyzed the properties of an M-deficient RV mutant. We provide evidence that M completely covers the ribonucleoprotein (RNP) coil and keeps it in a condensed form. As determined by cosedimentation experiments, not only the M-RNP complex but also M alone was found to interact specifically with the glycoprotein G. In contrast, an interaction of G with the nucleoprotein N or M-less RNP was not observed. In the absence of M, infectious particles were mainly cell associated and the yield of cell-free infectious virus was reduced by as much as 500,000-fold, demonstrating the crucial role of M in virus budding. Supernatants from cells infected with the M-deficient RV did not contain the typical bullet-shaped rhabdovirus particles but instead contained long, rod-shaped virions, demonstrating severe impairment of the virus formation process. Complementation with M protein expressed from plasmids rescued rhabdovirus formation. These results demonstrate the pivotal role of M protein in condensing and targeting the RNP to the plasma membrane as well as in incorporation of G protein into budding virions.

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Rabies virus matrix protein targets host actin cytoskeleton: A Protein-Protein interaction analysis

Pathogens and Disease ◽

10.1093/femspd/ftaa075 ◽

2020 ◽

Author(s):

Fatemeh Zandi ◽

Vahid Khalaj ◽

Fatemeh Goshadrou ◽

Anna Meyfour ◽

Alireza Gholami ◽

...

Keyword(s):

Rabies Virus ◽

Matrix Protein ◽

Molecular Mechanisms ◽

Interaction Analysis ◽

Super Resolution ◽

M Protein ◽

Docking Simulations ◽

Cytoskeleton Organization ◽

Rat Brainstem ◽

Rabies Pathogenesis

Abstract Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris (P. pastoris) and was partially purified. Subsequently, 2D-Far-WB determined six rat brainstem proteins interacted with recombinant M protein which were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process, and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation (Co-IP), was only successful for M-actin cytoplasmic1 interaction. Totally, our study revealed actin cytoplasmic1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

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Preparation and characterisation of monoclonal antibodies against the N protein of the SHpd/2012 strain of porcine epidemic diarrhoea virus

Veterinární Medicína ◽

10.17221/23/2018-vetmed ◽

2018 ◽

Vol 63 (No. 10) ◽

pp. 468-475 ◽

Cited By ~ 1

Author(s):

SJ Ding ◽

YR Luo ◽

ST Zhou ◽

C. Xie ◽

K. Wang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Western Blotting ◽

Test Strip ◽

Diagnostic Methods ◽

Economic Losses ◽

Hybridoma Cells ◽

N Protein ◽

Porcine Epidemic Diarrhoea Virus ◽

Diarrhoea Virus ◽

And Control

Porcine epidemic diarrhoea is caused by the porcine epidemic diarrhoea virus, and is a highly contagious disease which affects the intestines of new-born piglets resulting in intense diarrhoea. Historically, the virus has caused enormous economic losses in the pig industry. In particular, the emergence of new epidemic strains means there is a pressing need for prevention and control of the disease. Owing to the specificity of the monoclonal antibodies now available, study of the pathogenesis, immune mechanisms and new diagnostic methods can be performed. In this study, 13 strains of positive hybridoma cells were prepared by immunising mice with purified whole porcine epidemic diarrhoea virus, and analysis was performed using ELISA and Western blotting. Three cell strains specifically recognised the porcine epidemic diarrhoea virus nucleocapsid protein (N protein). In this study, we report the characterisation of effective tools for the establishment of porcine epidemic diarrhoea virus diagnostic methods and we have specifically generated primary antibodies for ELISA, IFA, test strip and Western blotting.

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Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649601 ◽

1993 ◽

Vol 70 (03) ◽

pp. 438-442 ◽

Cited By ~ 6

Author(s):

B Grøn ◽

C Filion-Myklebust ◽

S Bjørnsen ◽

P Haidaris ◽

F Brosstad

Keyword(s):

Molecular Weight ◽

Monoclonal Antibodies ◽

Human Plasma ◽

Western Blotting ◽

Isoelectric Point ◽

Factor Xiii ◽

Cross Linking ◽

2D Electrophoresis ◽

Complex Phenomenon ◽

Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

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Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies.

Journal of Virology ◽

10.1128/jvi.49.1.248-252.1984 ◽

1984 ◽

Vol 49 (1) ◽

pp. 248-252 ◽

Cited By ~ 26

Author(s):

K L van Wyke ◽

J W Yewdell ◽

L J Reck ◽

B R Murphy

Keyword(s):

Monoclonal Antibodies ◽

Influenza A Virus ◽

Influenza A ◽

Matrix Protein ◽

Antigenic Characterization

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5352584 Monoclonal antibodies which bind (E)-5-(2-bromovinyl)-arabinofuranosyluracil and diagnostic methods based thereon

Biotechnology Advances ◽

10.1016/0734-9750(96)84964-0 ◽

1995 ◽

Vol 13 (4) ◽

pp. 798

Keyword(s):

Monoclonal Antibodies ◽

Diagnostic Methods

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Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

International Journal of Molecular Sciences ◽

10.3390/ijms22063166 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3166

Author(s):

Jwala Priyadarsini Sivaccumar ◽

Antonio Leonardi ◽

Emanuela Iaccarino ◽

Giusy Corvino ◽

Luca Sanguigno ◽

...

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Western Blotting ◽

Cancer Biomarkers ◽

Trypsin Digestion ◽

Protein Fragments ◽

Target Epitope ◽

Potential Activity ◽

Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

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Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways

Journal of Virology ◽

10.1128/jvi.00761-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9897-9906 ◽

Cited By ~ 19

Author(s):

Florence Larrous ◽

Alireza Gholami ◽

Shahul Mouhamad ◽

Jérôme Estaquier ◽

Hervé Bourhy

Keyword(s):

Cell Death ◽

Amino Acid ◽

Matrix Protein ◽

Full Length ◽

M Protein ◽

Genotype 3 ◽

Cell Death Pathways ◽

Acid Fragment ◽

M Proteins ◽

Cellular Apoptosis

ABSTRACT The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

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Visual detection of granulocyte surface antigens using the avidin-biotin complex.

Journal of Histochemistry & Cytochemistry ◽

10.1177/32.7.6376617 ◽

1984 ◽

Vol 32 (7) ◽

pp. 712-716 ◽

Cited By ~ 7

Author(s):

M Henke ◽

L M Yonemoto ◽

G S Lazar ◽

L Gaidulis ◽

T Hecht ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Indirect Immunofluorescence ◽

Visual Detection ◽

Hla Antigens ◽

Surface Antigens ◽

Cell Populations ◽

Surface Markers ◽

Visual Test ◽

Avidin Biotin Complex

A visual test for detection of granulocyte surface markers using the avidin-biotin complex (ABC) has been developed. That this assay is highly specific, reproducible, and sensitive was determined by studying the expression of HLA antigens on granulocytes with monoclonal antibodies. Further, using granulocyte specific alloantisera, the results of the ABC test compared well to data from leukoagglutination assays and indirect immunofluorescence tests. The assay is particularly advantageous in that granulocytes can be stored, only small amounts of cells and sera are needed, and heterogeneous cell populations can easily be studied.

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