Le 1er transfert de brin, étape cruciale de la transcription inverse impliquant la protéine de nucléocapside du VIH-1 (NC), repose sur l’appariement de la séquence r de l’ADN « strong stop » (ADNss) avec la séquence 3’ R de l’ARN viral (3’UTR) qui forme les tiges-boucles TAR et polyA. La séquence r est supposé former les tiges-boucles cTAR et cpolyA. Le transfert repose donc probablement sur l’hybridation de molécules structurées. La structure secondaire de l’ADNss n’a jamais été déterminée. L’objectif a été d’identifier les interactions et structures gouvernant l’hybridation de l’ADNss avec l’ARN 3’UTR. Les outils de la biologie moléculaire et trois sondes de structure ciblant l’ADN ont été utilisés pour atteindre cet objectif. Nos résultats sont les suivants : 1) l’ADN cTAR nu se replie sous la forme de deux conformations différentes qui sont en équilibre ; 2) la NC peut déplacer l’équilibre vers l’une des conformations et se fixer préférentiellement sur la boucle interne du cTAR ; 3) la NC est exigée pour former un hétéroduplex constitué de l’intégralité de l’ADNss et du 3’ UTR ; 4) l’hybridation ADNss-3’UTR peut être initiée à partir de plusieurs sites dans 0,2 mM MgCl2 ; 5) l’ADNss forme deux conformations en équilibre dans 0,2 mM MgCl2 et principalement une seule dans 2 mM MgCl2 ; 6) dans l’ADNss, la NC se fixe préférentiellement au niveau de la région simple-brin qui relie les tiges-boucles cTAR et cpolyA. Cette fixation joue probablement un rôle important dans l’hybridation des tiges-boucles ARN et ADN complémentaires. Notre étude permet de mieux comprendre la transcription inverse et la recombinaison qui dépend du transfert de brin interne. / The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3’ R sequence of viral RNA (3’ UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3’ UTR; 4) the annealing of ssDNA to 3’ UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-05-12T13:41:13Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Maureen_Fernandes.pdf: 925477 bytes, checksum: adc4b50d524df10be1448912c874f299 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-12T21:22:55Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Maureen_Fernandes.pdf: 925477 bytes, checksum: adc4b50d524df10be1448912c874f299 (MD5) / Made available in DSpace on 2017-05-12T21:22:55Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Maureen_Fernandes.pdf: 925477 bytes, checksum: adc4b50d524df10be1448912c874f299 (MD5) Previous issue date: 2015-12-03 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS / O objetivo deste trabalho foi a expressão da região conservada da proteína do nucleocapsídeo do vírus da cinomose (aa 222-316) em Escherichia coli e posterior avaliação de suas propriedades antigênicas e imunogênicas. Para tal, a região conservada do gene da proteína do nucleocapsídeo do vírus da cinomose canina (NP CDV) foi selecionada e codón otimizada para expressão em E. coli. O gene sintético foi inserido em um vetor pAE e clonado em cepas E. coli TOP10F. A proteína recombinante foi expressa em E. coli cepa Star e purificada por cromatografia de afinidade. A concentração de proteína obtida após purificação foi aproximadamente 300 mg/mL. A expressão da proteína recombinante NP do CDV (rCDVNP) foi confirmada por eletroforese em gel de poliacrilamida (SDS-PAGE) e Western blot utilizando anticorpos monoclonais anti His tag. A antigenicidade da rCDVNP foi demonstrada por Western blot e ELISA indireto, empregando soros de cães positivos e negativos para cinomose canina. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (IgY) foi purificada a partir da gema de ovo, utilizando o método de delipidação com água destilada acidificada e precipitação com sulfato de amônia. A purificação de IgY foi confirmada por SDS-PAGE e a concentração média de IgY que se obteve foi 28,55 mg/mL. A produção de IgY anti-rCDVNP foi analisada por Western blot e ELISA indireto. Os resultados demonstraram que o gene sintético codificante da região conservada da NP do CDV foi clonado com sucesso utilizando o vetor pAE em E. coli, resultando posteriormente na expressão de porção da NP na forma recombinante. A rCDVNP demonstrou ser antigênica, uma vez que os soros de cães positivos para cinomose canina reconheceram a proteína. A rCDVNP foi imunogênica após inoculação em galinhas, sendo possível o isolamento e purificação de alta concentração de IgY específica a partir de gema de ovo. Estes resultados sugerem que tanto a rCDVNP quanto a IgY anti-rCDVNP produzida podem ser utilizadas como ferramentas úteis em ensaios de imunodiagnóstico da cinomose canina. / The purpose of this study was the expression of the conserved region of Canine distemper virus nucleocapsid protein (aa 222-316) in Escherichia coli and subsequent evaluation of its antigenic and immunogenic properties. The conserved region of Canine distemper virus nucleocapsid (CDV NP) gene was selected and codon optimized for E. coli expression. The synthetic gene was inserted in a pAE vector and cloned into E. coli TOP10F strain. The recombinant protein was expressed in E. coli Star strain and purified by affinity chromatography. The protein yield after purification was approximately 300 mg/mL. Recombinant CDV NP (rCDVNP) expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis by using His-Tag monoclonal antibodies. The rCDVNP antigenicity was demonstrated by Western blot and ELISA, employing positive and negative CDV dog sera. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was isolated and purified from egg yolk, according the delipidation method with acidified distilled water and the precipitation method with ammonium sulfate. The purification of IgY was confirmed by SDS-PAGE and the mean yield of IgY was 28.55 mg/mL. The production of IgY anti-rCDVNP was analyzed by Western blot and ELISA assays. The results showed that the synthetic gene coding the conserved region of the NP CDV was successfully cloned using pAE vector in E. coli, resulting in the expression of a recombinant NP. The rCDVNP proved to be antigenic, since distemper positive dog sera recognized the protein. The rCDVNP proved to be immunogenic after inoculation in hens and it was possible to isolate and purify high concentration of specific IgY from egg yolk. These results suggest that both rCDVNP as IgY anti-rCDVNP produced can be used as useful tools in immunodiagnostic assays of canine distemper.

<p>The human coronavirus NL63 was discovered in 2004 by a team of researchers in Amsterdam. Since its discovery it has been shown to have worldwide spread and affects mainly children, aged 0-5 years old, the immunocompromised and the elderly. Infection with HCoV-NL63 commonly results in mild upper respiratory tract infections and presents as the common cold, with symptoms including fever, cough, sore throat and rhinorrhoea. Lower respiratory tract findings are less common but may develop into more serious complications including bronchiolitis, pneumonia and croup. The primary function of the HCoV-NL63 nucleocapsid (N) protein is the formation of theprotective ribonucleocapsid core. For this particle to assemble, the N-protein undergoes N-N dimerization and then interacts with viral RNA. Besides the primary structural role of the Nprotein, it is also understood to be involved in viral RNA transcription, translation and replication, including several other physiological functions. The N-protein is also highly antigenic and elicits a strong immune response in infected patients. For this reason the N-protein may serve as a target for the development of diagnostic assays. We have used bioinformatic analysis to analyze the HCoV-NL63 N-protein and compared it to coronavirus N-homologues. This bioinformatic analysis provided the data to generate recombinant clones for expression in a bacterial system. We constructed recombinant clones of the N-protein of SARS-CoV and HCoV-NL63 and synthesized truncated clones corresponding to the N- and C-terminal of the HCoV-NL63 N-protein. These heterologously expressed proteins will serve the basis for several post-expression studies including characterizing the immunogenic epitope of the N-protein as well identifying any antibody crossreactivity between coronavirus species.</p>

The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3' R sequence of viral RNA (3' UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3' UTR; 4) the annealing of ssDNA to 3' UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination.

<p>The human coronavirus NL63 was discovered in 2004 by a team of researchers in Amsterdam. Since its discovery it has been shown to have worldwide spread and affects mainly children, aged 0-5 years old, the immunocompromised and the elderly. Infection with HCoV-NL63 commonly results in mild upper respiratory tract infections and presents as the common cold, with symptoms including fever, cough, sore throat and rhinorrhoea. Lower respiratory tract findings are less common but may develop into more serious complications including bronchiolitis, pneumonia and croup. The primary function of the HCoV-NL63 nucleocapsid (N) protein is the formation of theprotective ribonucleocapsid core. For this particle to assemble, the N-protein undergoes N-N dimerization and then interacts with viral RNA. Besides the primary structural role of the Nprotein, it is also understood to be involved in viral RNA transcription, translation and replication, including several other physiological functions. The N-protein is also highly antigenic and elicits a strong immune response in infected patients. For this reason the N-protein may serve as a target for the development of diagnostic assays. We have used bioinformatic analysis to analyze the HCoV-NL63 N-protein and compared it to coronavirus N-homologues. This bioinformatic analysis provided the data to generate recombinant clones for expression in a bacterial system. We constructed recombinant clones of the N-protein of SARS-CoV and HCoV-NL63 and synthesized truncated clones corresponding to the N- and C-terminal of the HCoV-NL63 N-protein. These heterologously expressed proteins will serve the basis for several post-expression studies including characterizing the immunogenic epitope of the N-protein as well identifying any antibody crossreactivity between coronavirus species.</p>

Abstract Yellow head virus (YHV) has caused mass mortalities in Penaeus monodon shrimp farmed throughout Southeast Asia since it was first discovered in the early 1990’s. YHV possesses a positive-sense, single-stranded RNA genome and a rod-shaped enveloped virion. Together with the closely related gill-associated virus (GAV) identified in P. monodon shrimp in Australia, it is classified in the genus Okavirus, family Roniviridae within the order Nidovirales. YHV particles contain only three structural proteins, a nucleocapsid (N) protein (p20) protein and two envelope glycoproteins gp116 and gp64. In this study, the glycosylation status of gp116 and gp64 extracted from YHV virions was characterized in detail, including the identification of active N-linked glycosylation sites and the nature of the attached carbohydrates. This was achieved by optimizing and applying a combination of methods that included SDS-PAGE followed by carbohydrate-specific staining of gels or probing of membrane-bound proteins using lectins with different carbohydrate specificities, enzymatic removal of N-linked carbohydrates and a variety of mass spectrometry techniques. In these analyses, it was found that N-linked glycans are the major contributor to the higher estimated mass of gp116 and gp64 by SDS-PAGE compared to those estimated from their deduced amino acid sequences. Neither gp116 nor gp64 were found to posses O-linked glycans. Mannose residues were identified to be the major glycan component of carbohydrates linked to gp116 and gp64 and are possibly the sole component of carbohydrate linked to gp64. Unlike gp64, other glycans such as terminal N-acetyl--D-galactosamine and N-acetyl--D-glucosamine were identified to be attached to gp116. Assuming that glycosylation processes in shrimp mimic those of vertebrates that are known in more detail, the nature of the glycans attached to gp116 suggests that they might be added and modified during the transportation of the protein from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN). Mass spectrometry analyses of tryptic peptides derived from the native glycoproteins and following their enzymatic deglycosylation, generated approximately 81% (gp116) and 66% (gp64) coverage of their predicted amino acid sequences. Detailed mass spectrometry analyses of peptides derived from the deglycosylated proteins identified that most of the potential N-linked glycosylated site in the virion envelope glycoproteins, 6 of 7 present in gp116 and 3 of 4 present in gp64 were identified to be modified by glycans. In gp116, one site was not identified and in gp64 one site was not utilized. As phosphorylation has been shown to affect nucleocapsid protein (N) functioning in vertebrate nidoviruses, SDS-PAGE using two phosphoprotein-specific staining methods, as well as mass spectrometry methods, were employed to examine whether the YHV N protein present in virions is phosphorylated. The protein staining methods provided contradicting results and no phosphate-containing peptides were identified by mass spectrometry. The apparent absence of phosphate in the N protein was also supported by its isoelectric point (pI ~10) determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) analysis, which was very similar to that predicted (pI = 9.98) from its deduced amino acid sequence. Taken together, the data suggest that the YHV N protein encapsulated within virions is not phosphorylated. The RNA-binding capability of the GAV N protein was assessed using an electrophoretic mobility shift assay (EMSA) technique. Full-length and variously truncated forms of the GAV N protein expressed in bacteria were assessed in the assays. It was found that the full-length recombinant N protein bound to RNA in a sequence non-specific manner. Analysis of the five truncated N protein constructs localized the RNA-binding domain to a 50 amino acid sequence in the N-terminal region residing between Met11 and Arg60. A motif rich in proline and arginine residues, which are commonly found in other RNA-binding proteins, occurred in first 18 amino acids of this region. Although RNA-binding was not sequence-specific, the data suggest that this region of the GAV N protein is the most likely site at which it interacts with and nucleates viral genomic RNA during nucleocapsid formation. A synthetic peptide spanning the 18 amino acid of the putative RNA-binding domain was shown to possess RNA-binding properties similar to the recombinant protein fragment. These results indicated that the 18 amino acid, proline and arginine rich motif (MPVRRPLPPQPPRNARLI) in the N-terminal region of the GAV N protein confers its RNA-binding function. Using an immuno-co-precipitation assay, a host protein was found to interact abundantly with the GAV N protein in infected lymphoid organ cells. Mass spectrometry analysis identified the protein as -actin. Immuno-histochemistical double-labeling methods in conjunction with observations made using confocal and electron microscopy revealed that actin and the N protein were co-located in cytoplasm of infected cells. Electron microscopy suggested that interaction of the two proteins occurs before nucleocapsid envelopment within virions, suggesting that -actin might be involved in transporting the N protein or the nucleocapsid from their sites of synthesis to the rough endoplasmic reticulum where the virion acquires its envelopes. In summary, the research described in this thesis has advanced understanding of the YHV/GAV proteome through the identification of the glycosylation sites in the envelope glycoproteins gp116 and gp64, and demonstrating that nucleocapsid protein encapsulated within virion is unlikely to be phosphorylated. Functional studies have also shown that the nucleocapsid protein binds RNA non-specifically through an 18 amino acid domain near its N-terminus and that it binds and co-localizes with -actin in infected cells, suggesting that -actin may play role in trafficking N protein in infected cells.

La tri-thérapie utilisée pour le traitement du VIH-1 est efficace mais limitée par l'apparition de résistances. Par conséquent, des cibles virales alternatives sont nécessaires. Une des cibles les plus prometteuses est la protéine nucléocapside (NC), qui est hautement conservée et qui joue un rôle essentiel dans le cycle viral. Dans ce contexte, le projet européen THINPAD a eu pour but de développer des inhibiteurs de la NC en combinant plusieurs approches : criblage virtuel, criblage secondaire in vitro, tests antiviraux et de toxicité. Pour le criblage in vitro, nous avons utilisé le test de déstabilisation de cTAR, hautement spécifique de l’activité chaperonne de NC. Cinq séries de molécules ont été sélectionnées par les premiers criblages et tests antiviraux. Après des études de relation structure-activité, une seule des cinq séries a été poursuivie jusqu’aux tests d'efficacité chez les souris. Les composés de cette série présentent une activité antivirale à des concentrations nanomolaires, mais ne sont pas actifs dans le modèle murin. Les études de mécanisme d'action ont révélés que leur activité antivirale était bien consécutive au ciblage de la NC. / Highly Active Anti-Retroviral Therapy (HAART) is successfully used for HIV-1 treatment, but is hampered by the appearance of drug resistance. Thereby, alternative drug targets are required. One of the most promising target is the nucleocapsid protein (NC), which is highly conserved and plays essential role in HIV life cycle. In this context, the European project THINPAD was organized with the aim to develop NC inhibitors. To fulfil this objective, several approaches were used, including virtual screening, in vitro secondary screening, in cellulo antivirals tests, and toxicity evaluation. For in vitro screening, the specific NC-promoted cTAR destabilization assay was used. Five series of molecules were selected by the first screenings and antiviral tests. After structure-activity relationship studies, only one series was continued until efficacy testing in mice. The compounds of this series exhibit antiviral activity at nanomolar concentrations but are not active in the murine model. The mechanisms of action studies revealed that their antiviral activity was indeed consecutive to the targeting of the NC.

Orientador: Hélio José Montassier / Banca: Camillo Del Cistia Andrade / Banca: Janete Apparecida Desidério Sena. / Resumo: Anticorpos monoclonais se constituem na base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por "phage display" contra a estirpe vacinal (H120) do VBI, foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01, IBVPR05, isoladas de surtos a campo no Brasil e SE-17, isolada nos Estados Unidos. Após três ciclos de "panning", foi identificado pelo ELISA um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que três desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzido em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus e no estudo de evolução de variantes desse vírus. / Abstract: Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of outbreaks of infectious bronchitis, because these antibodies are homogeneous, highly specific and fully characterized, allowing the improvement of detection of immunological techniques and antigenic characterization of avian infectious bronchitis virus strains (IBV). We used a phage display library prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments reacting with heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning a set of 15 scFv antibodies was expressed in phages and exhibited crossreaction in ELISA with these three viral strains. Western-blotting analysis showed that three of this clone set were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41 strain of IBV. In conclusion, the recombinant fragments of monoclonal antibodies expressed by phage-display technique have a great potential for future use in immunodiagnostic techniques and study the evolution of variant strains of this virus. / Mestre

>Magister Scientiae - MSc / Acute respiratory infections (ARI) continue to be the leading cause of acute illnesses worldwide and remain the most important cause of infant and young children mortality. Many viruses such as rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, adenoviruses and coronaviruses are deemed to be the etiological agents responsible for ARI’s in children. The recently discovered coronaviruses HCoV-HKU1 and HCoV-NL63 contribute significantly to the hospitalization of children with ARI’s. HCoV-NL63 was first identified in 2004, as the pathogen responsible for the hospitalization of a 7 month old child presenting with coryza, conjunctivitis and fever. Since then a significant amount of knowledge has been gained in the clinical spectrum on this virus, however HCoV-NL63 is still not well characterized on the molecular and proteomic level. This dissertation focuses on bringing about this characterization by cloning the HCoV-NL63 Nucleocapsid gene to be expressed in a bacterial system and transfecting the Nucleocapsid, Membrane and Envelope genes into a Mammalian cell culture system in order for its respective proteins to be expressed. With the use of Bioinformatic analytic tools certain characteristics of HCoV-NL63 Nucleocapsid, Membrane and Envelope proteins are able to be identified, as well as certain motifs and/or regions that are important in the functioning of these proteins. By comparing the results obtained for HCoV-NL63 N,M and E to other well studied coronavirus homologous will enlighten us on the potential role(s) of these proteins in determining HCoV-NL63 pathogenicity and infectivity. vi Although certain functions of these proteins can be deduced by the means of bioinformatics analysis, it is still imperative for it to be extensively characterized In Vitro. This will therefore form a fundamental step in the development of many other projects, which unfortunately fall outside the scope of this M.Sc thesis.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The human coronavirus NL63 was discovered in 2004 by a team of researchers in Amsterdam. Since its discovery it has been shown to have worldwide spread and affects mainly children, aged 0-5 years old, the immunocompromised and the elderly. Infection with HCoV-NL63 commonly results in mild upper respiratory tract infections and presents as the common cold, with symptoms including fever, cough, sore throat and rhinorrhoea. Lower respiratory tract findings are less common but may develop into more serious complications including bronchiolitis, pneumonia and croup. The primary function of the HCoV-NL63 nucleocapsid (N) protein is the formation of theprotective ribonucleocapsid core. For this particle to assemble, the N-protein undergoes N-N dimerization and then interacts with viral RNA. Besides the primary structural role of the Nprotein, it is also understood to be involved in viral RNA transcription, translation and replication, including several other physiological functions. The N-protein is also highly antigenic and elicits a strong immune response in infected patients. For this reason the N-protein may serve as a target for the development of diagnostic assays. We have used bioinformatic analysis to analyze the HCoV-NL63 N-protein and compared it to coronavirus N-homologues. This bioinformatic analysis provided the data to generate recombinant clones for expression in a bacterial system. We constructed recombinant clones of the N-protein of SARS-CoV and HCoV-NL63 and synthesized truncated clones corresponding to the N- and C-terminal of the HCoV-NL63 N-protein. These heterologously expressed proteins will serve the basis for several post-expression studies including characterizing the immunogenic epitope of the N-protein as well identifying any antibody crossreactivity between coronavirus species. / South Africa