scholarly journals Immunogenicity and crossreactivity of antibodies to SARS-CoV-2 nucleocapsid protein

2020 ◽  
Author(s):  
Carlota Dobaño ◽  
Rebeca Santano ◽  
Alfons Jiménez ◽  
Marta Vidal ◽  
Jordi Chi ◽  
...  
Keyword(s):  
Common Cold ◽  
Sequence Homology ◽  
Nucleocapsid Protein ◽  
Full Length ◽  
Primary Target ◽  
N Protein ◽  
Sequence Identity ◽  
C Terminus ◽  
Minimal Sequence ◽  
Human Coronaviruses

ABSTRACTCOVID-19 patients elicit strong responses to the nucleocapsid (N) protein of SARS-CoV-2 but binding antibodies are also detected in prepandemic individuals, indicating potential crossreactivity with common cold human coronaviruses (HCoV) and questioning its utility in seroprevalence studies. We investigated the immunogenicity of the full-length and shorter fragments of the SARS-CoV-2 N protein, and the crossreactivity of antibodies with HCoV. We indentified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognised N229E N, and IgGs to HKU1 N recognised SARS-CoV-2 N. Crossreactivity with SARS-CoV-2 was stronger for alpha-rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition. Higher preexisting IgG to OC43 N correlated with lower IgG to SARS-CoV-2 in rRT-PCR negative individuals, reflecting less exposure and indicating a potential protective association. Antibodies to SARS-CoV-2 N were higher in patients with more severe and longer symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N is a primary target of SARS-CoV-2-specific and HCoV crossreactive antibodies, both of which may affect the acquisition of immunity to COVID-19.

scholarly journals Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

2020 ◽  
Author(s):  
Corinne A. Lutomski ◽  
Tarick J. El-Baba ◽  
Jani R. Bolla ◽  
Carol V. Robinson
Keyword(s):  
Antibody Response ◽  
Nucleocapsid Protein ◽  
Electron Transfer Dissociation ◽  
Rna Binding ◽  
Vaccine Development ◽  
Cyclophilin A ◽  
Proteolytic Processing ◽  
Full Length ◽  
Oligomeric State ◽  
N Protein

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

scholarly journals Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

2001 ◽  
Vol 8 (3) ◽  
pp. 598-603 ◽  
Author(s):  
Sarah Wootton ◽  
Gabriella Koljesar ◽  
Liuzhan Yang ◽  
Kyoung-Jin Yoon ◽  
Dongwan Yoo
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
Nucleocapsid Protein ◽  
Conformational Epitope ◽  
Single Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Beta Strand ◽  
N Protein ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.

scholarly journals Antibody Response against the SARS-CoV-2 Nucleocapsid Protein and Its Subdomains—Identification of Pre-Immunization Status by Human Coronaviruses with Multipanel Nucleocapsid Fragment Immunoblotting

COVID ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 105-114
Author(s):  
Sahra Pajenda ◽  
Sebastian Kapps ◽  
Thomas Reiter ◽  
Raimundo Freire ◽  
Veronique A. J. Smits ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Posterior Part ◽  
Rapid Identification ◽  
Diagnostic Methods ◽  
Serum Samples ◽  
N Protein ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Global Pandemic ◽  
Human Coronaviruses

A novel beta coronavirus that emerged in late December 2019 triggered a global pandemic. Diagnostic methods for rapid identification of infected individuals were established in new biotechnological approaches. Vaccine production and application to individuals and measurement of SARS-CoV-2 antibodies also began. Serum samples from 240 health care workers were collected at three-month intervals over nine months. Indirect SARS-CoV-2 nucleocapsid IgG ELISA tests were used to identify humoral immune responses. All seropositive individuals and those with borderline ELISA values were tested with a specifically generated multipanel nucleocapsid fragment immunoblot. Of the 240 individuals, 24 showed seroconversion in ELISA after experiencing COVID-19. All of them showed a positive reaction against the full-length nucleocapsid protein in the immunoblot. The highest reactivity was seen either against fragment N(100–300) or in a minority against the posterior part N(200–419). In general, the staining pattern of COVID-19 patients showed four phenotypes. In contrast, three individuals classified as borderline by ELISA reacted exclusively with fragments N(1–220) and N(100–300) containing the octamer amino acid sequence FYYLGTGP, which is identical in human coronaviruses sharing this sequence with SARS-CoV-2. These represent a unique and thus fifth phenotype. This work suggests the existence of distinct phenotypic patterns of IgG production towards N-protein subdomains.

scholarly journals Antigenicity Analysis of Different Regions of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

2004 ◽  
Vol 50 (6) ◽  
pp. 988-995 ◽  
Author(s):  
Zeliang Chen ◽  
Decui Pei ◽  
Lingxiao Jiang ◽  
Yajun Song ◽  
Jin Wang ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nucleocapsid Protein ◽  
Vaccine Candidate ◽  
Protein Microarray ◽  
Full Length ◽  
Protein Fragments ◽  
N Protein ◽  
Conformational Epitopes ◽  
Glass Slides ◽  
Linear Epitopes

Abstract Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein. Methods: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli. After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments. Results: The full-length protein and two other fragments reacted with all 52 sera tested. Four important regions with possible epitopes were identified and named as EP1 (amino acids 51–71), EP2 (134–208), EP3 (249–273), and EP4 (349–422), respectively. EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera. EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested. Conclusion: The N protein is a highly immunogenic protein of the SARS-CoV. Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH2 terminus. The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV.

Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

1993 ◽  
Vol 70 (03) ◽  
pp. 454-457 ◽  
Author(s):  
Claus Bregengaard ◽  
Ole Nordfang ◽  
Per Østergaard ◽  
Jens G L Petersen ◽  
Giorgio Meyn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Fold Increase ◽  
Volume Of Distribution ◽  
Full Length ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

scholarly journals Persistent COVID-19 Symptoms Minimally Impact the Development of SARS-CoV-2-Specific T Cell Immunity

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 916
Author(s):  
Hengsheng Fang ◽  
Adam D. Wegman ◽  
Kianna Ripich ◽  
Heather Friberg ◽  
Jeffrey R. Currier ◽  
...  
Keyword(s):  
Cellular Immunity ◽  
Viral Infections ◽  
T Cell Immunity ◽  
N Protein ◽  
Cell Immunity ◽  
Public Health Challenge ◽  
Human Coronaviruses ◽  
Cellular Immune ◽  
Persistent Viral Infections ◽  
Insight Into

SARS-CoV-2 represents an unprecedented public health challenge. While the majority of SARS-CoV-2-infected individuals with mild-to-moderate COVID-19 resolve their infection with few complications, some individuals experience prolonged symptoms lasting for weeks after initial diagnosis. Persistent viral infections are commonly accompanied by immunologic dysregulation, but it is unclear if persistent COVID-19 impacts the development of virus-specific cellular immunity. To this end, we analyzed SARS-CoV-2-specific cellular immunity in convalescent COVID-19 patients who experienced eight days or fewer of COVID-19 symptoms or symptoms persisting for 18 days or more. We observed that persistent COVID-19 symptoms were not associated with the development of an overtly dysregulated cellular immune response. Furthermore, we observed that reactivity against the N protein from SARS-CoV-2 correlates with the amount of reactivity against the seasonal human coronaviruses 229E and NL63. These results provide insight into the processes that regulate the development of cellular immunity against SARS-CoV-2 and related human coronaviruses.

scholarly journals Immunological imprinting of the antibody response in COVID-19 patients

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Teresa Aydillo ◽  
Alexander Rombauts ◽  
Daniel Stadlbauer ◽  
Sadaf Aslam ◽  
Gabriela Abelenda-Alonso ◽  
...  
Keyword(s):  
Immune Response ◽  
Severe Acute Respiratory Syndrome ◽  
Antibody Response ◽  
Humoral Immune Response ◽  
Nucleocapsid Protein ◽  
Spike Protein ◽  
Ongoing Research ◽  
Variable Regions ◽  
Humoral Immune ◽  
Human Coronaviruses

AbstractIn addition to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humans are also susceptible to six other coronaviruses, for which consecutive exposures to antigenically related and divergent seasonal coronaviruses are frequent. Despite the prevalence of COVID-19 pandemic and ongoing research, the nature of the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here we longitudinally profile the early humoral immune response against SARS-CoV-2 in hospitalized coronavirus disease 2019 (COVID-19) patients and quantify levels of pre-existing immunity to OC43, HKU1 and 229E seasonal coronaviruses, and find a strong back-boosting effect to conserved but not variable regions of OC43 and HKU1 betacoronaviruses spike protein. However, such antibody memory boost to human coronaviruses negatively correlates with the induction of IgG and IgM against SARS-CoV-2 spike and nucleocapsid protein. Our findings thus provide evidence of immunological imprinting by previous seasonal coronavirus infections that can potentially modulate the antibody profile to SARS-CoV-2 infection.

scholarly journals SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 530
Author(s):  
Soo Jin Oh ◽  
Ok Sarah Shin
Keyword(s):  
Nucleocapsid Protein ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Antiviral Immune Response ◽  
N Protein ◽  
Nonstructural Protein 1 ◽  
Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

scholarly journals Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

2000 ◽  
Vol 74 (19) ◽  
pp. 9028-9038 ◽  
Author(s):  
J.-B. Nousbaum ◽  
S. J. Polyak ◽  
S. C. Ray ◽  
D. G. Sullivan ◽  
A. M. Larson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Antiviral Therapy ◽  
Variable Region ◽  
Response To Therapy ◽  
Full Length ◽  
Variable Regions ◽  
C Terminus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

scholarly journals Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

2004 ◽  
Vol 78 (15) ◽  
pp. 8281-8288 ◽  
Author(s):  
M. A. Mir ◽  
A. T. Panganiban
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Viruses ◽  
Sin Nombre Virus ◽  
Viral Capsid ◽  
Genomic Rna ◽  
Rna Molecules ◽  
N Protein ◽  
High Affinity ◽  
Negative Sense

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

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