Production of measles nucleoprotein in different expression systems and its use as a diagnostic reagent

Warnes, Alan; Fooks, Anthony R.; Stephenson, John

doi:10.1016/0166-0934(94)90141-4

Cited by 32 publications

(15 citation statements)

References 10 publications

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“…McCarthy and Lazzarini [1982] also observed multiple bands of MW 54±56 kDa, 45 kDa and 39 kDa in CsCl gradient puri®ed mumps NP from mumps virus infected Vero cells and suggested that the virion NP protein contains protease-sensitive sites. Similar degradation attributed to protease activity was observed by Warnes et al [1994] for measles rNP produced in three different expression systems and in measles virus infected cells and by Hummel et al [1992] for the measles rNP expressed in insect cells using recombinant baculovirus at high multiplicity of infection. Despite the degradation of puri®ed rNP observed in immunoblots, the cleavage of rNP protein does not necessarily affect its ability to form or remain in nucleocapsid-like structure suggesting that strong intra or inter molecular, non-covalent bonds within the structure maintain the conformation of the protein.…”

Section: Discussionsupporting

confidence: 68%

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology

Samuel

Sasnauskas²,

Jin

et al. 2001

Journal of Medical Virology

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To develop improved reagents for mumps serology a high-level yeast expression system was employed to produce recombinant mumps nucleoprotein (rNP). The rNP was purified by CsCl gradient centrifugation and yielded approximately 15 mg/l of yeast culture. Electron microscopy of the rNP revealed characteristic herring-bone structures. The electrophoretic mobility of rNP in yeast cells was similar to native NP in SDS-PAGE. Monoclonal antibodies to rNP reacted with native mumps virus nucleoprotein by immunofluorescence assay. A monoclonal antibody to native mumps virus NP reacted with rNP by Western blot assay. The rNP was investigated as antigen in an IgM capture enzyme immunoassay (EIA) using a horseradish peroxidase conjugate of monoclonal antibody to the rNP. Eighteen sera previously found to be positive by IgM capture radioimmunoassay (MACRIA) and 30 sera that were mumps IgM negative by MACRIA were tested by mumps IgM capture EIA. The results for the two test were concordant. In addition, 26 rheumatoid factor positive sera and 35 sera that were IgM positive for measles, rubella or parvovirus B19 were tested. Fifty-nine sera were negative by mumps IgM capture EIA but two sera collected from two infants 3 and 6 weeks after mumps, measles and rubella vaccination were positive. Mumps MACRIA confirmed these results. Compared to MACRIA the overall sensitivity was 100% (20/20) and specificity was 96.8% (30/31). The yeast expressed rNP was highly immunogenic and suitable for use in IgM capture EIA for the diagnosis of mumps.

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Section: Discussionsupporting

confidence: 68%

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology

Samuel

Sasnauskas²,

Jin

et al. 2001

Journal of Medical Virology

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“…RNA is tightly bound within the nucleocapsid, which is not believed to dissociate during RNA synthesis. Expression of the N protein from measles virus and mumps virus in a variety of heterologous systems results in the formation of nucleocapsid-like particles (3,26,44,46,47,52,53). For the measles virus N protein, it has been shown that these structures result from the nonspecific encapsidation of cellular RNA (47), a result which is likely to hold for the mumps virus N protein as well, based on the reported densities of mumps virus nucleocapsid-like particles in CsCl (44,46).…”

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confidence: 99%

Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

Kingston

Baase

2004

J Virol

100

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We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly ␣-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K d ) was determined to be 13 M at 20°C and 35 M at 37°C. Our data are consistent with a model in which an ␣-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

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“…Viral ELISA antigens were produced in primary avian fibroblasts as described previously and monoclonal antibodies were derived and purified as described by Stephenson et al (1984). ELISA was performed and titres calculated as described previously (Warnes et al, 1994).…”

Section: Infection Of Cells With a Recombinant Adenovirus Containing mentioning

confidence: 99%

The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell

Crooks

Lee

Easterbrook

et al. 1994

Journal of General Virology

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Production of measles nucleoprotein in different expression systems and its use as a diagnostic reagent

Cited by 32 publications

References 10 publications

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology

Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell

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