Antigenic and Immunogenic Investigation of B-Cell Epitopes in the Nucleocapsid Protein of Peste des Petits Ruminants Virus

ABSTRACT Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.

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Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.4.658-664.2004 ◽

2004 ◽

Vol 11 (4) ◽

pp. 658-664 ◽

Cited By ~ 11

Author(s):

Kang-Seuk Choi ◽

Jin-Ju Nah ◽

Young-Joon Ko ◽

Shien-Young Kang ◽

Kyoung-Jin Yoon ◽

...

Keyword(s):

Antigenic Site ◽

Enzyme Linked Immunosorbent Assay ◽

Carboxy Terminus ◽

Rinderpest Virus ◽

N Protein ◽

Amino Terminal ◽

Immunogenic Epitope ◽

Carboxy Terminal ◽

Gst Fusion Proteins

ABSTRACT The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N1-525), an amino-terminal construct (N1-179), and a carboxy-terminal construct (N414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N414-496 was much more antigenic than GST-N1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues 440VPQVRKETRASSR452 (site 1), 479PEADTDPL486 (site 2), and 520DKDLL524 (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.

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THE DISTRIBUTION OF ANTIGENIC DETERMINANTS IN RAT SKIN COLLAGEN

Journal of Experimental Medicine ◽

10.1084/jem.133.6.1309 ◽

1971 ◽

Vol 133 (6) ◽

pp. 1309-1324 ◽

Cited By ~ 38

Author(s):

Herbert Lindsley ◽

Mart Mannik ◽

Paul Bornstein

Keyword(s):

Terminal Region ◽

Antigenic Determinants ◽

Collagen Molecule ◽

Rat Skin ◽

Amino Terminal ◽

Skin Collagen ◽

Native Collagen ◽

Carboxy Terminal Region ◽

Carboxy Terminal ◽

Hyperimmune Rabbit

Immunological studies of rat skin collagen were carried out with a sensitive and quantitative radioimmunoassay. Hyperimmune rabbit antisera to rat skin collagen and isolated α2 chains were used. Iodine-labeled α chains and CNBr-produced peptides served as test antigens, and native collagen, α chains, and CNBr peptides were employed as inhibitors in the assay. The α1 and α2 chains were immunologically distinct. Although the α1 chain was not immunogenic, antibodies to α1 were detected in antisera to the intact collagen molecule. The major antigenic determinant of the α1 chain was located in α1-CB6 which constitutes the carboxy-terminal region of the chain. The α2 chain contained two non-cross-reacting antigenic determinants, one in the amino-terminal region (α2-CB1) and the other in the carboxy-terminal region (α2-CB5) of the chain. The native collagen molecule was less effective than isolated α chains in inhibiting binding of labeled peptides to antisera, indicating that antigenic determinants were less accessible in the triple helical molecule. These immunologic studies are consistent with preliminary comparative biochemical data which indicate that interspecies structural differences in collagen predominate at both the amino- and carboxy-terminal ends of the chains.

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A Major Determinant for Membrane Protein Interaction Localizes to the Carboxy-Terminal Domain of the Mouse Coronavirus Nucleocapsid Protein

Journal of Virology ◽

10.1128/jvi.79.21.13285-13297.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13285-13297 ◽

Cited By ~ 66

Author(s):

Kelley R. Hurst ◽

Lili Kuo ◽

Cheri A. Koetzner ◽

Rong Ye ◽

Bilan Hsue ◽

...

Keyword(s):

Viral Envelope ◽

M Protein ◽

N Protein ◽

Amino Terminal ◽

Terminal Domain ◽

Dominant Negative Mutation ◽

Carboxy Terminal Domain ◽

Domain 3 ◽

Positive Strand Rna ◽

Carboxy Terminal

ABSTRACT The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, MΔ2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MΔ2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of MΔ2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.

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Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein

Journal of Virology ◽

10.1128/jvi.00440-09 ◽

2009 ◽

Vol 83 (14) ◽

pp. 7221-7234 ◽

Cited By ~ 71

Author(s):

Kelley R. Hurst ◽

Cheri A. Koetzner ◽

Paul S. Masters

Keyword(s):

Nucleocapsid Protein ◽

Fluorescent Protein ◽

Hepatitis Virus ◽

M Protein ◽

N Protein ◽

Terminal Domain ◽

Domain 3 ◽

Positive Strand Rna ◽

Carboxy Terminal

ABSTRACT The coronavirus nucleocapsid protein (N), together with the large, positive-strand RNA viral genome, forms a helically symmetric nucleocapsid. This ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (M), which is the principal constituent of the virion envelope. Previous work with the prototype coronavirus mouse hepatitis virus (MHV) has shown that a major determinant of the N-M interaction maps to the carboxy-terminal domain 3 of the N protein. To explore other domain interactions of the MHV N protein, we expressed a series of segments of the MHV N protein as fusions with green fluorescent protein (GFP) during the course of viral infection. We found that two of these GFP-N-domain fusion proteins were selectively packaged into virions as the result of tight binding to the N protein in the viral nucleocapsid, in a manner that did not involve association with either M protein or RNA. The nature of each type of binding was further explored through genetic analysis. Our results defined two strongly interacting regions of the N protein. One is the same domain 3 that is critical for M protein recognition during assembly. The other is domain N1b, which corresponds to the N-terminal domain that has been structurally characterized in detail for two other coronaviruses, infectious bronchitis virus and the severe acute respiratory syndrome coronavirus.

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Purification and characterization of Haemophilus influenzae pili, and their structural and serological relatedness to Escherichia coli P and mannose-sensitive pili.

Journal of Experimental Medicine ◽

10.1084/jem.161.1.145 ◽

1985 ◽

Vol 161 (1) ◽

pp. 145-159 ◽

Cited By ~ 13

Author(s):

N G Guerina ◽

S Langermann ◽

G K Schoolnik ◽

T W Kessler ◽

D A Goldmann

Keyword(s):

Haemophilus Influenzae ◽

Cross Reactivity ◽

Coli Strain ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Cell Agglutination ◽

E Coli ◽

Amino Terminal ◽

Multiple Copies

Haemophilus influenzae pili were purified, and their physical and serological properties were examined. The solution properties of the pili were determined, and then a purification scheme involving repeated cycles of precipitation and solubilization was developed. The purified pili from one type b isolate (A02) were found to consist of multiple copies of a 25,000 mol wt subunit. Amino-terminal sequence analysis of A02 pili was carried out to 40 amino acid residues, and a remarkable degree of sequence homology was found with E. coli P and mannose-sensitive (MS) pili (27.5 and 25% homology, respectively). Purified A02 pili were found to be highly immunogenic, and serological analysis by enzyme-linked immunosorbent assay and whole piliated cell agglutination revealed significant cross-reactivity between A02 pilus antiserum and the pili of seven other H. influenzae strains tested (heterologous titers = 2-100% of the homologous titer). Cross-reactivity was also observed between the H. influenzae pili (five of eight strains tested) and the P pili from E. coli strains HU849 and 3669; no cross-reactivity was detected with MS pili from E. coli strain H10407 and C94. The structural similarities between H. influenzae and E. coli P and MS pili suggest a common gene ancestry.

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In-House Immunofluorescence Assay for Detection of SARS-CoV-2 Antigens in Cells from Nasopharyngeal Swabs as a Diagnostic Method for COVID-19

Diagnostics ◽

10.3390/diagnostics11122346 ◽

2021 ◽

Vol 11 (12) ◽

pp. 2346

Author(s):

Athene Hoi-Ying Lam ◽

Jian-Piao Cai ◽

Ka-Yi Leung ◽

Ricky-Ruiqi Zhang ◽

Danlei Liu ◽

...

Keyword(s):

Epithelial Cells ◽

Nucleocapsid Protein ◽

Diagnostic Method ◽

Polyclonal Antibodies ◽

Respiratory Viruses ◽

Cross Reactivity ◽

Immunofluorescence Assay ◽

Respiratory Epithelial Cells ◽

N Protein ◽

Respiratory Epithelial

Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.

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The optimization of an ultra-sensitive single molecule assay for the multiplexed detection of SARS-CoV-2 antigens

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.309 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S145-S145

Author(s):

Y Senussi ◽

Z N Swank ◽

D R Walt

Keyword(s):

Single Molecule ◽

Respiratory Infections ◽

Sample Volume ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Plasma Samples ◽

N Protein ◽

Children And Young Adults ◽

Inflammatory Syndrome

Abstract Introduction/Objective SARS-CoV-2 antigens, including the nucleocapsid (N) protein, spike protein, and its S1 subunit have served as key biomarkers for research and diagnostic purposes. We previously developed quantitative single molecule array (Simoa) assays to measure the concentration of spike, S1 subunit and N protein in plasma samples with femtomolar limits of detection. We aimed to test antibodies that were not available early in the pandemic, reduce assay cross-reactivity, develop a multiplexed assay for spike, S1, and N protein in order to minimize the sample volume needed. Methods/Case Report Using the Simoa platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 17 S1 subunit and spike antibodies for a total of 130 antibody-pair combinations, we performed dilution linearity experiments to determine the ideal dilution factor, spike and recovery experiments, tested the assay using S1 subunit from other human coronavirus HKV1, NL63, and 229E, pre-pandemic plasma samples from patients that were sick with viral or bacterial respiratory infections. We then used the best antibody pairs to measure S1 and spike in plasma samples collected from patients with severe SARS-CoV-2. Lastly, we conjugated the best-performing capture antibodies for spike, S1 and N to beads labeled with different fluorophores to test if the assay for all three antigens could be multiplexed. Results (if a Case Study enter NA) We observed no cross-reactivity with S1 from other coronavirus strains, no detection of S1 or spike in a cohort of 30 pre-pandemic samples and successfully developed a multiplexed assay for the detection of spike, S1, and N protein, enabling us to use 50% less sample volume. Conclusion Reduction of necessary sample volume is important for studies involving multisystem inflammatory syndrome in children (MIS-C), and possible adverse effects of SARS-CoV-2 vaccinations on children and young adults. An improved assay with minimal cross-reactivity will also be useful to study individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

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The Carboxy Terminal Region on Spike Protein of Porcine Epidemic Diarrhea Virus (PEDV) Is Important for Evaluating Neutralizing Activity

Pathogens ◽

10.3390/pathogens10060683 ◽

2021 ◽

Vol 10 (6) ◽

pp. 683

Author(s):

Ki-Jong Kang ◽

Dong-Hwan Kim ◽

Eui-Ju Hong ◽

Hyun-Jin Shin

Keyword(s):

Vaccine Efficacy ◽

Terminal Region ◽

Enzyme Linked Immunosorbent Assay ◽

Neutralizing Epitope ◽

Nucleocapsid Proteins ◽

S Protein ◽

Neutralizing Activity ◽

Diarrhea Virus ◽

Carboxy Terminal Region ◽

Carboxy Terminal

In this study, we evaluated 62 sow sera samples from PED-vaccinated sows to compare the serum neutralizing test (SNT) and enzyme-linked immunosorbent assay (ELISA). We performed protein ELISA (pELISA) using fragments of spike proteins S1, S2, S3 and entire nucleocapsid proteins, and found a correlation between the SNT and ELISA in PEDV-vaccinated sera. Sera with higher neutralizing activity showed higher titers of IgG. In the antibody profiling, the neutralizing activities are correlated with the levels of the spike antibody, especially the S3 region. We confirmed that the carboxy-terminal region, including the endodomain of the S protein, induced stronger neutralizing activity than the ectodomain. This region of the S protein could be useful for evaluating PED vaccine efficacy, and it is a strong neutralizing epitope of PEDV. The S3 protein could be useful for evaluating PED vaccine efficacy, and it is a strong neutralizing epitope of PEDV.

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Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

Journal of Virology ◽

10.1128/jvi.75.23.11851-11862.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11851-11862 ◽

Cited By ~ 28

Author(s):

S. Michael Rothenberg ◽

Mari N. Olsen ◽

Louise Chang Laurent ◽

Rachel Adams Crowley ◽

Patrick O. Brown

Keyword(s):

Cell Surface ◽

Receptor Binding ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Receptor Binding Domain ◽

Amino Terminal ◽

Carboxy Terminal Region ◽

Env Protein ◽

Carboxy Terminal ◽

Murine Leukemia

ABSTRACT The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

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Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

Journal of Virology ◽

10.1128/jvi.03212-15 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4357-4368 ◽

Cited By ~ 27

Author(s):

Lili Kuo ◽

Kelley R. Hurst-Hess ◽

Cheri A. Koetzner ◽

Paul S. Masters

Keyword(s):

Nucleocapsid Protein ◽

Hepatitis Virus ◽

Virus Assembly ◽

Mouse Hepatitis Virus ◽

M Protein ◽

Growth Defect ◽

S Protein ◽

N Protein ◽

Protein Incorporation ◽

Carboxy Terminal

ABSTRACTThe coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought.IMPORTANCEThe assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

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