Purification and Characterization of Nipah Virus Nucleocapsid Protein Produced in Insect Cells

Abstract

The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A 260 /A 280 ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.Nipah virus (NiV), the etiologic agent for both human and swine diseases, was first isolated from the cerebrospinal fluid of human patients and classified in the family Paramyxoviridae (7). Members of this family are nonsegmented, negativestranded RNA viruses composed of helical nucleocapsids enclosed within an envelope to form roughly spherical and pleomorphic particles (21). There are two subfamilies within the family Paramyxoviridae: the Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae has been divided into three genera: Rubulavirus, Respirovirus, and Morbillivirus. Recently, a fourth genus, Henipavirus, has been proposed, which includes the Hendra and Nipah viruses (25).Nucleocapsids of paramyxoviruses appear as typical helical or herringbone-like structures (12) which are often used for the identification of viruses in this family. The helical parameters have been studied extensively by negative staining and metal shadowing electron microscopy (3,8,16). Paramyxovirus nucleocapsid (N) proteins with masses ranging between 58 and 60 kDa consist of a conserved N-terminal domain that constitutes three-quarters of the protein and a nonconserved, acidic C-terminal domain. The presence of the charged C-terminal domain of N protein seems to be connected with the loose coiling of the nucleocapsids and the observed variation in helical parameters, since removal of this domain by trypsin (26) or preparation of nucleocapsids in high salt (14) stiffens the structure, making it more regular.There is a need for rapid detection as well as serological diagnosis o...

Development of a neutralization assay for Nipah virus using pseudotype particles

“…Expression systems were used to produce antigens for enzyme immunoassays including the NiV N protein (Chen et al, 2006; Yu et al, 2006), a truncated phosphoprotein antigen (Chen et al, 2007), or the glycoproteins of NiV (Eshaghi et al, 2005a; Eshaghi et al, 2004; Eshaghi et al, 2005b). Other assays described the use of monoclonal antibodies for immunohistochemical based diagnosis (Tanimura et al, 2004; Xiao et al, 2008), or a modified plaque assay in which the plates are prepared in BSL-4 and inactivated by gamma irradiation before staining at BSL-2 (Crameri et al, 2002).…”

The emergence of Nipah virus, a highly pathogenic paramyxovirus

“…[59][60][61] Recently developed ELISAs now utilize recombinant-expressed purified NiV antigens, and a number of high-affinity monoclonal antibodies have been generated for diagnostic and prophylactic purposes. [62][63][64][65][66][67][68][69][70][71][72][73] While serum neutralization tests (SNTs) have long been a reference standard, the next generation of SNTs will circumvent the use of Plasmid-based expression of NiV P, V, and W proteins from both Malaysian and Bangladesh strains inhibit transcription of firefly luciferase from an interferon stimulated response element (ISRE) promoter. 293 cells were transfected for 48 h with individual plasmids expressing NiV P, V, W, or C proteins along with ISRE-firefly luciferase and control Renilla luciferase plasmid, and then stimulated with interferon (IFN) beta for 6 h before harvest.…”

Serodiagnosis Using Recombinant Nipah Virus Nucleocapsid Protein Expressed in Escherichia coli

“…Eshaghi, Tan, and colleagues reported that Nipah virus N (NiV-N) protein expressed in insect cells and Escherichia coli assembled into different types of structures of different lengths, including spherical, ring-like, and herringbone-like particles, under electron microscopy and that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition (7,16,20). But since only a limited number of swine samples were tested, more studies are required to assess the use of the recombinant N protein in routine diagnosis, especially for human samples.…”