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Journal of Clinical Microbiology, July 2005, p. 3172-3177, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3172-3177.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Majid Eshaghi,¹ Wen Siang Tan,^1,2 Swee Tin Ong,¹ and Khatijah Yusoff^1,2*

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,¹ Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia²

Received 1 October 2004/ Returned for modification 5 November 2004/ Accepted 7 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 A₂₆₀/A₂₈₀ 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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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, negative-stranded 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 of the virus to determine the seroprevalence of anti-NiV in selected population groups, particularly those individuals who are in close contact with fruit bats, in the high-risk area, and in probable future outbreaks. Currently, production of immunological reagents for these assays requires the highest level of microbiological security (biohazard level 4), which is limited to only a few laboratories around the world. Recombinant DNA technology provides an alternative means for the production of safer diagnostic reagents. Since the NiV N protein is a major immunogen, we set out to express this protein in a baculovirus expression system to produce the antigen for an immunoassay as well as to facilitate structural and functional analyses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viruses and antisera. Inactivated NiV from infected cell culture medium and swine antisera with known serum neutralizing test titers (SN50) were obtained from the Veterinary Research Institute, Ipoh, Malaysia. The serum samples were collected during the 1998 to 1999 outbreak of NiV in Malaysia.

Cloning, expression, and purification of the N protein of NiV. Fig. 1 shows the strategy for amplification and cloning of the NiV N gene in the pFastBacHT vector. Competent Escherichia coli DH10BAC cells, containing bacmid (baculovirus shuttle vector plasmid) and helper plasmid, were used to generate recombinant bacmids according to the manufacturer's (BAC-TO-BAC baculovirus expression system; Life Technologies) instructions.

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FIG. 1. Strategy for amplification and cloning of the NiV N gene. Extracted viral RNA (vRNA) (using TRIZOL LS reagent; Life Technologies) was used as a template for cDNA synthesis using the Superscript II RNaseH (–) reverse transcriptase (RT; Life Technologies), which was subsequently used in a PCR amplification using the Platinum high-fidelity Taq DNA polymerase (Life Technologies). rTEV, recombinant tobacco etchvirus protease cleavage site.

The recombinant bacmid DNA was transfected into insect cells using the CELLFECTIN reagent. Spodoptera frugiperda (Sf9) cells were cultured at 27°C in Sf-900 II SFM. All cell culture media and reagents were purchased from Life Technologies. For each transfection, 9 x 10⁵ cells were seeded in 35-mm wells of a six-well plate and allowed to attach for 1 h. Lipid reagent and DNA were diluted separately into 100 µl of Sf-900 II SFM cells and then combined to form lipid-DNA complexes, which were then diluted to 1 ml with SFM and added to the cells. The cells were incubated for 5 h at 27°C after which the transfection medium was removed and replaced with fresh medium. These cells were analyzed for protein expression at 24 to 72 h posttransfection. Viral supernatant was collected at 72 h posttransfection.

Control or infected cells were washed with phosphate-buffered saline (10 mM sodium phosphate, 0.15 M NaCl, pH 7.5) and lysed directly in 62.5 mM Tris-HCl (pH 6.8) containing 2% sodium dodecyl sulfate (SDS) and boiled for 10 min. Cell extracts were cleared by centrifugation, and samples were analyzed on 12% polyacrylamide gels. Proteins in the lysates of recombinant virus inoculated with Sf9 cells were analyzed by Western blotting as described by Sambrook and Russell (28). Broad-range protein markers (Gibco-BRL) were used in SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analyses. Swine anti-NiV polyclonal antibodies (1/500 dilution) were used as the primary antibodies. Appropriate species-specific immunoglobulin conjugated to alkaline phosphatase (1/5,000 dilution) was used as the secondary antibody.

The recombinant N protein fused to a histidine affinity tag at its N terminus was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin as recommended by the manufacturer (BAC-TO-BAC baculovirus expression system; Life Technologies). Briefly, Sf9 cells at a density of about 1.5 x 10⁶/ml in shaker flasks were infected with the recombinant baculovirus at a multiplicity of infection (MOI) of about 5 and incubated with shaking for 72 h at 27°C. The infected cells were harvested by centrifugation at 500 x g for 5 min at 4°C. The pellet was resuspended in lysis buffer (50 mM Tris-HCl [pH 8.5], 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 1% Tween 20). After incubation for 15 min at 4°C on a rotating shaker, the preparation was clarified by centrifugation at 30,000 x g for 15 min. The recombinant protein was precipitated with 10% ammonium sulfate saturation and dialyzed overnight against Tris buffer (50 mM Tris-HCl, pH 8.5) with 4 changes of buffer. The supernatant was loaded onto an Ni-NTA column, equilibrated with buffer A (20 mM Tris-HCl [pH 8.5], 5 mM 2-mercaptoethanol, 500 mM KCl, 10% [vol/vol] glycerol, 20 mM imidazole). The column was washed successively with 10 ml of buffer A, 5 ml of buffer B (20 mM Tris-HCl [pH 8.5], 5 mM 2-mercaptoethanol, 1 M KCl, 10% [vol/vol] glycerol, 20 mM imidazole), and finally, 5 ml of buffer A again. Protein was eluted from the column with 5 ml of buffer C (20 mM Tris-HCl [pH 8.5], 5 mM 2-mercaptoethanol, 100 mM KCl, 10% [vol/vol] glycerol, imidazole [increment concentrations from 100 mM to 500 mM]). The samples were collected in 0.5-ml fractions and analyzed by SDS-PAGE and Western blotting. Protein concentrations were determined according to the Bradford method (5) using bovine serum albumin (Sigma) as the standard.

Electron microscopy. Recombinant N protein (20 µl) purified from the nickel column was absorbed to carbon-coated grids and stained with 2% uranyl acetate. The grids were viewed under an energy filter transmission electron microscope (LEO 912AB).

Dynamic light scattering and spectroscopy. The polydispersity of the sample was studied with a dynamic light-scattering instrument (DynaPro-MS/X; Proterion, Inc., United Kingdom) at 20°C. The concentration and spectrum of the samples were measured using the Anthelie Advanced spectrophotometer. The buoyant density of the sample was calculated as reported previously (11).

ELISA. The titer of the anti-N polyclonal antibodies in swine sera was determined by an indirect enzyme-linked immunosorbent assay (ELISA). All washing steps were carried out three times with TTB (0.05% Tween 20 in Tris-buffered saline [TBS]). All antigen and antibody dilutions were in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Flat-bottom polystyrene microtiter plates were used as solid-phase adsorbents (Techno Plastic Products Immunomax high-binding flat-bottom ELISA plate). The recombinant N protein in TBS (100 ng/well; 100 µl) or inactivated NiV (100 µl) was added to the wells. After incubation for 18 h at room temperature (RT), the plates were washed and then blocked with 200 µl of SEA BLOCK blocking buffer (Pierce) containing chicken serum (1/100) for 1 h at RT. Subsequently, the plates were washed three times with TTB and incubated for 2 h at RT with the appropriate dilution (1/20) of the swine sera from infected and noninfected animals. After washing with TTB, the appropriate dilution (1/3,000) of anti-swine immunoglobulin G (IgG) alkaline phosphates (KPL) was added and the microtiter plates were incubated further for 1 h at RT. Following another washing step, the enzyme substrate solution containing 1 mg/ml p-nitrophenyl phosphate (Sigma) in 0.1 M diethanolamine (Sigma), pH 10.3, was added. The reaction was stopped after 30 min of incubation at RT, and the A₄₀₅ values were measured with a microtiter plate reader (Bio-Rad). The experiment was repeated as described above for the detection of the NiV-specific IgM antibodies, except that anti-swine IgM (1/5,000) conjugated with horseradish peroxidase (BETHYL) was used as the secondary antibody and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt] with 0.005% hydrogen peroxide was used as the substrate. The sensitivity and specificity of the test were calculated as [a/(a + c) x 100] and [d/(b + d) x 100], respectively, where a is the number of true positives, b is the number of false positives, c is the number of false negatives, and d is the number of true negatives. The positive and negative predictive values were calculated as [a/(a + b)] and [d/(c + d)], respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A recombinant baculovirus was generated using the transposon-mediated insertion of the corresponding genetic construct under the polyhedron promoter of the Autographa californica nuclear polyhedrosis virus (22). The Sf9 cells were transfected with this recombinant bacmid DNA. Following transfection, the supernatant was harvested and the recombinant bacmid was propagated at a MOI of 1 to get a higher titer of recombinant bacmid. In the second infection, an MOI of 5 was used to infect the cells to express the N protein. Total cellular extracts of Sf9 cells that had been infected with baculovirus were separated by SDS-PAGE (Fig. 2A). Western blot analysis, using a pooled anti-NiV serum, revealed a major band of approximately 62 kDa, which was absent in the lysate of Sf9 cells as well as in those infected with wild-type baculovirus (Fig. 2B). The molecular mass of this band corresponds to that of the expected fusion protein. There was no expression immediately after transfection, but the level of expression increased gradually after each of the subsequent two infections (data not shown).

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FIG. 2. The NiV N protein expressed by the recombinant baculovirus in insect cells. SDS-PAGE (A) and Western blot (B) analysis of Sf9 cells infected with wild-type baculovirus (lanes 1), noninfected Sf9 cells (lanes 2), and Sf9 cells infected with recombinant baculovirus (lanes 3). Arrows indicate the expected bands. Lanes M, molecular mass markers.

A time course study was performed to ascertain the optimal time for obtaining the highest yield of the recombinant N protein. To do this, the Sf9 cells were infected with the recombinant baculovirus at an MOI of 5 and sampling was done every 24 h postinfection for 4 days. At each time point, cells were counted and their viabilities were determined by trypan blue exclusion staining. The cells were pelleted, and their recombinant protein contents were determined by Western blotting and densitometric scanning of the reactive bands. It was found that the highest level of expression could be achieved after 3 days of incubation (data not shown). Approximately 2 to 3 mg/ml of the recombinant protein was obtained from 1.5 x 10⁶ infected Sf9 cells.

The dialyzed N protein was purified through an Ni-NTA resin column after ammonium sulfate precipitation. After washing with a low concentration of imidazole (20 mM), unbound and weakly bound proteins were removed. The polyhistidine-tagged N protein (His-N) was finally eluted from the column by increasing the imidazole concentration to 500 mM. SDS-PAGE analysis and Coomassie brilliant blue staining of the eluted fractions showed that the His-N protein was efficiently bound to the column and that the corresponding fractions contained highly purified protein (Fig. 3A). Western blot analysis with anti-NiV sera proved the presence of N protein (Fig. 3B). The concentration of the purified recombinant N protein from the total infected cell proteins was estimated to be approximately 200 to 300 µg/ml.

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FIG. 3. Characterization of purified NiV N protein expressed in insect cells (lanes 1) by SDS-PAGE (A) and Western blot analysis (B). Lanes M, molecular mass markers.

The absorption spectrum of the purified protein had an optimum absorption at 260 nm, indicating the presence of nucleic acids. RNase treatment of the purified protein reduced the absorption significantly, whereas DNase treatment did not. The A₂₆₀/A₂₈₀ ratio of the recombinant NiV N-RNA was found to be 1.1 ± 0.05 (result from three independent samples). This suggests that recombinant NiV N protein binds to cellular RNA in a nonspecific manner.

Electron microscopic analysis of the purified His-N protein produced in baculovirus showed spherical and ring- and herringbone-like structures (Fig. 4). The purified sample was polydispersed, and polymodal regression analysis indicated the sample contained 96% small particles and 4% large particles. The buoyant density of the sample was found to be 1.08 ± 0.02 g/ml (result from four independent samples).

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FIG. 4. Transmission electron micrographs showing formation of full and empty spherical structures (A) and ring- and herringbone-like structures (B) in purified NiV His-N protein. Bars, 100 nm (A) and 50 nm (B).

Sixty-five swine sera assessed independently by serum neutralizing titer (SN50) (39 positives and 26 negatives) were analyzed using purified recombinant N protein and inactivated NiV for the detection of NiV-specific IgG and IgM antibodies in swine sera by ELISA (Table 1 and Fig. 5). Using the recombinant N protein and inactivated NiV IgG ELISAs, 39 of the 65 serum samples were shown to be positive and 26 serum samples were negative, similar to the results obtained by SN50. However, of these 65 serum samples, 44 were positive and 21 were negative when they were tested by recombinant N antigen and inactivated NiV IgM ELISAs.

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TABLE 1 . Comparison of results from SN50 with results obtained by ELISA based on inactivated NiV and recombinant N protein

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FIG. 5. Comparative analysis of reactivity of NiV-specific IgG (A and C) and IgM (B and D) antibodies to recombinant N protein (A and B) and inactivated NiV (C and D) in an indirect ELISA. The error bars represent standard deviations from the means of the results from triplicate determinations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, Nipah virus N fusion protein (His-N, 62 kDa) was produced in insect cells, where it associates with the cellular nucleic acids. This suggests that the N protein of NiV has nonspecific binding activities with the host nucleic acids, similar to those observed for rabies, measles, and Marburg virus N proteins expressed in insect cells (17, 24).

From a structural point of view, the His-N protein purified by nickel affinity chromatography with the conditions used gave rise to a diversity of spherical forms, from full to empty particles and ring- and herringbone-like structures, when they were observed under an electron microscope. The diameter of the small particles (8 to 12 nm) determined by electron microscopy is in good agreement with the hydrodynamic radius measured with dynamic light scattering. These were, however, smaller than the size of the nucleocapsids of NiV (18 to 21 nm). This is most probably due to the existence of the different genomic RNAs or different conformations. Most recently, the expression of the N fusion protein in E. coli was found to form herringbone-like subnucleocapsid particles (30), but no ring-like structures were observed.

Earlier studies have shown that the folding of the N protein of negative-strand RNA viruses into its mature form is dependent upon the presence of the viral genome (2, 4), although the formation of the herringbone- and ring-like subnucleocapsid structures in the absence of viral RNA has also been reported for proteins of other paramyxoviruses, such as respiratory syncytial virus in E. coli (27), measles virus expressed in insect cells and E. coli (13, 32), Newcastle disease virus expressed in both insect cells and E. coli (10, 19, 20), and Sendai virus expressed in mammalian cells (6). It is believed that the spherical structure formation in this study can either be due to the different stages of protein maturation, where the spherical forms are probably the immature types or due to incorrect maturation of the protein. To the best of our knowledge, this study is the first to show that a paramyxovirus N protein assembles into spherical particles.

The NiV N protein produced in E. coli is mainly found in inclusion bodies (30). The solubility of the recombinant protein can be improved by expression under the control of different promoters and hosts, truncating the protein, and changing the temperature of the growing culture. On the contrary, the majority of the NiV N protein expressed in insect cells was in the soluble fraction.

A breakdown product of approximately 51 kDa was routinely observed for His-N protein, which could be due to degradation of the C-terminal end of the His-N protein. Thus, this observation is in good agreement with other paramyxovirus N proteins, in which the C termini are susceptible to proteolytic removal (14, 15, 26, 29, 30).

In this study, the potential diagnostic utility of the purified recombinant His-N protein has been explored, and it is clear that the antigen facilitates the detection of antibodies in swine naturally infected with NiV. The N proteins of negative-strand RNA viruses are highly immunogenic in nature and have been used as antigens for diagnostic purposes, including the N protein of rabies (18), measles (31), vesicular stomatitis virus (1), and Newcastle disease virus (9). It has also been reported that N protein-based immunoassays can be used to monitor vaccination programs and as a diagnostic test in differentiating between vaccinated and infected animals in conjunction with subunit vaccines (23). When ELISAs based on inactivated NiV and recombinant N protein were used to detect NiV-specific IgM and IgG antibodies, titers of IgM did not correspond with IgG titers, as was expected. Six of the samples known to be negative by SNT and also confirmed by IgG ELISA were found to have NiV-specific IgM antibodies using IgM ELISA. Our result may suggest that IgM ELISA is necessary as a complementary test for IgG ELISA and SN50. The use of IgM and IgG ELISAs in tandem facilitates detailed analysis of the antibody responses in NiV infection. The ELISA results obtained based on recombinant N protein showed high sensitivity and specificity, suggesting a potential alternative to conventional ELISA methods using inactivated whole virus.

It should be noted that more studies are needed to assess the use of the recombinant His-N protein in routine diagnosis, since a limited number of field samples was tested. Here, Western blot analysis and ELISA suggest that the recombinant His-N protein exhibits the epitopes and conformation necessary for specific antigen-antibody recognition. Based on this study, it should now be possible to use His-N protein for further structure and function analysis of the N protein and to develop an immunoassay for detection of the NiV antibodies.

 
We thank the Veterinary Research Institute (VRI) of Malaysia, especially Sharifah Syed Hassan, for generously providing the inactivated NiV swine isolate, swine anti-NiV sera, and SN50 results.

This study was supported by grant no. 26-02-03-0128 from the Ministry of Science, Technology, and Innovation, Malaysia (MOSTI). M.E. is supported by a National Science Fellowship (NSF) from MOSTI.

 
* Corresponding author. Mailing address: Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM 43400, Serdang, Selangor, Malaysia. Phone: 603 89466701. Fax: 603 89430913. E-mail: kyusoff{at}putra.upm.edu.my.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, July 2005, p. 3172-3177, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3172-3177.2005
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