Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins

The nucleoprotein N is the most abundant viral component in the RVFV virion and also in virus infected cells. N is tightly associated with the three genomic RNA segments, forming the three nucleocapsids. N protein plays a number of roles that are essential in virus replication. In addition it also interacts with L, Gn and Gc, although the nature of their interactions have not yet been defined. In order to generate N protein in sufficient amount in the absence of other viral proteins we generated a recombinant baculovirus (as described in Methods) and examined the level of N protein expression in insect cells. Insect Sf9 cells were infected with this recombinant baculovirus for four days and the presence of N protein in the cell lysate was assessed by SDS-PAGE analysis. A strong extra band of 26 KDa equivalent to the expected size of the N protein was detected in the infected cell lysate (Fig. 1A, lane 2). This band was not present in the lysate from uninfected cells (Fig. 1A, lane 1). Western blot analysis using monoclonal antibody specific to RVFV N protein confirmed that the extra band was the RVFV N protein (Fig. 1A, lane 4).

Recent studies have demonstrated that the basic oligomeric status of N protein in purified ribonucleoprotein (RNP) from RVFV infected cells is a dimer, however it exhibited multimeric organization when RNPs were cross-linked with glutaraldehyde [12]. To investigate if recombinant N synthesized in insect cells is capable of oligomerisation, the supernatant of infected insect cells were clarified, ultracentrifuged through a sucrose cushion and protein products were analyzed by gel filtration column chromatography. The products obtained from the gel filtration are shown in Fig. 1B. A distinct protein peak was detected in the exclusion region (the column exclusion size limit was 1300 kD) suggesting that the N protein was able to form complex structures. To determine the position of N protein complex a series of protein control molecular markers were included and their relative position is indicated in the figure. When aliquots of gel filtration fractions were analyzed by SDS-PAGE (Fig. 1C), a band of the expected size for N was detected, suggesting that N was the major component in those fractions. To confirm further that the eluted band was indeed the N protein of RVFV, and had the same mobility with the N protein band of the cell lysate an aliquot was analyzed by Western blot using anti-N antibody (Fig. 1D, lane 3).

To determine if N protein containing fractions could form any particulate complex structure, fractions containing N protein were clarified by ultracentrifugation and aliquots were visualized by electron microscopy (EM). Distinct particulate structures could be detected under EM (Fig. 1E). The size of these structures ranged from 56 to 78 nm, suggesting that N could indeed form complex multimeric structures.

RVFV virus particle are enveloped, and the two structural glycoproteins Gn and Gc are inserted in the membrane that surrounds the RNP. To investigate if RVFV glycoproteins can be assembled together with N protein in baculovirus expression system, a dual protein expression vector was designed. Previous works using vaccinia and baculovirus systems have shown that the expression of Gn/Gc from the fourth AUG of M segment produce high level and correct processing of both proteins [24, 28]. Indeed of the five AUG initiation codons present in the upstream sequence of Gn only the fourth AUG is in optimal translation context sequence. Therefore for the baculovirus construct, the open reading frame of M segment from the fourth AUG was used. The Gn/Gc and N sequences were inserted into the baculovirus transfer vector under the control of two separate polyhedrin promoters. The recombinant baculovirus was generated as described in Methods.

Insect cells were infected with the recombinant baculovirus containing the three RVFV genes. After 3 days cells were lysed and the lysates were analyzed by SDS-PAGE followed by Commassie blue staining. While expression of N protein was at a high level and clearly visible, bands of Gc and Gn were not convincing (Fig. 2A, lane 2). Therefore a Western analysis using the appropriate antibodies was performed. An aliquot of cell lysate from uninfected cells and from baculovirus infected cells expressing β-Gal were also included as control. Only in samples from cells infected with recombinant baculovirus, proteins bands corresponding to Gn (Fig. 2B, lane 2) and Gc (Fig. 2B, lane 5) could be detected by specific antibodies against those proteins. This result confirmed that in addition to N protein, both Gn and Gc were also expressed and the Gc/Gn was properly processed to generate the proteins in the insect cells.

It has been reported previously that when RVFV Gn and Gc are expressed individually, Gn is targeted to the Golgi while Gc is retained in the ER [18, 19]. However, Filone et al. have recently shown that the overexpression of Gn and Gc by alphavirus replicon vectors resulted in the localization of these proteins in the cell surface [20]. Therefore it was of interest if this effect could be observed in insect cells by recombinant baculovirus expressing these glycoproteins. To visualize the expression of Gn/Gc complex on the cell surface, insect cells were infected with the recombinant baculovirus expressing Gn, Gc and N proteins and 30 hours post-infected cells were fixed and processed for immunofluorescence. Since these cells were not permeabilized only proteins expressed in the surface of cells should be detected. When specific antibody against Gn was used as a primary antibody and FITC-conjugated as secondary antibody, a strong signal around the surface of infected cells was easily visible (Fig. 2C, upper panel). Similar result was obtained when a specific antibody against Gc and TRITC-conjugated secondary antibody were used (Fig. 2D, upper right panel). As a control, cells were infected with a recombinant baculovirus expressing β-Galactosidase protein and processed similarly. Although low level of background was detected when the FITC-conjugated secondary antibody was used, no background was observed for the TRITC-conjugated antibody (Fig. 2C and 2D, lower panels).

Thus, the expression of RVFV Gn and Gc proteins in insect cells resulted in detection of both proteins on the surface of the non-permeabilized infected cells. The presence of Gn and Gc on the surface of infected insect cell suggests that the both proteins were correctly folded and properly processed.

It has been demonstrated that Gn and Gc are responsible for virus entry during natural infection using a class II fusion mechanism activated by low pH [21, 29]. More recently the cell-cell fusion activity was demonstrated for Gn and Gc proteins that were expressed on the surface of cells at high levels using alphavirus replicon vectors [20]. Therefore to determine if recombinant Gn and Gc proteins expressed in insect cells were functionally active, fusion of adjacent membranes was investigated. Sf9 cells were infected with the baculovirus expressing both Gn and Gc proteins as described above and 24 h post-infected cells were incubated for two hours with a monoclonal antibody against gp64, a baculovirus surface glycoprotein, in order to inhibit activity of gp64 that has ability to induce cell-cell fusion after low pH induction. Cell media were then shift to pH 5.0 for two minutes and regularly examined for syncytia formation. Large syncytia were observed in cells expressing Gn/Gc proteins after two hours of treatment at low pH but no evidence of fusion was detected in cells maintained at normal pH of 6.5 (compare Fig. 3A, upper panels). As a control cells infected with another recombinant baculovirus that expresses Bluetongue virus (BTV) outer capsid protein VP2 [30], a non-fusogenic protein was also included. No evidence of syncytia formation was observed in VP2 expressed control cells (Fig. 3A, lower panels). These results suggest that Gn/Gc complex was functionally active and was solely responsible for inducing adjacent membrane fusion after low pH treatment.

To further characterize the pH dependence of the fusion activity of the complex Gn/Gc a range of pH were tested. The fusogenic ability was assessed as the average number of syncytia in at least 20 fields of visual microscopy at 100× magnification, in three independent experiments. As shown in Fig. 3B a significant number of syncytia were counted when cells expressing Gn/Gc were treated at low pH (between pH 4.5 to 5.5) and as expected the number decrease at high pH (pH 6.0 to 7.0). As expected, no evidence of fusion was observed in control cells expressing BTV VP2. These results demonstrated that Gn/Gc expressed in insect cells by recombinant baculovirus has a pH dependent fusion activity. Similar result were observed with alphavirus expressed Gn/Gc [20].

These results suggest that Gn/Gc expressed in the baculovirus expression system is fully functional and share similar characteristics with that of native RVFV infection and other recombinant systems.

Since the recombinant N protein alone could initiate assembly of a particulate structure in insect cells it was likely that the expression of Gn and Gc together with the N protein may assemble as a particulate structure. To examine if the three expressed proteins could assemble into VLP, the supernatant from infected cells were collected after three days of infection. After clarification, the supernatant was loaded on to a 20% sucrose cushion and subjected to ultracentrifugation. The pellet was subsequently resuspended and further purified by ultracentrifugation through potassium tartrate-glycerol gradient and fractions were collected. Aliquots were analyzed by SDS-PAGE (data not shown) and those fractions with a band corresponding to N were concentrated by ultracentrifugation. The presence of Gn and N in the concentrated sample was detected by Western blot using monoclonal antibodies (Fig. 4A, lane 3 and 4), and all three proteins, N, Gn and Gc were also detected by polyclonal antibody against RVFV virus particles (Fig. 4A, lane 5).

In order to analyze if this concentrated sample indeed contained VLPs, an aliquot of the purified and concentrated fraction was examined by EM. Particulate structures with a spiky outer layer ranging from 90–120 nm were found in this fraction (Fig. 4B). These structures resemble the structure of RVFV. Some particles preserved nearly perfect surface subunits, which were presumably formed by Gn and Gc heterodimeric complex similar to that of virion particles [8] (Fig. 4B). The clarity of these surface spikes could easily be counted around 26 to 37 as shown in Fig. 4B (note the three particles in the lower panels). These results suggest that the structures purified from the supernatant of cells expressing RVFV structural proteins N and Gn/Gc are indeed VLPs.

Further, to determine the localization of VLPs in the cytosol and to confirm that VLPs were matured in the vacuoles, insect cells infected with the recombinant baculovirus expressing the three RVFV proteins were harvested, fixed and processed for ultra-section analysis. The results obtained from EM analysis showed that particulate structures, similar to RVFV virion particles, were released into vacuoles (Fig. 4C, indicated by black arrows). There were also a large number of inclusion bodies accumulated in the cytoplasm (Fig. 4D, indicated by arrow). Similar virion particles and inclusion bodies have also been reported to be present in RVFV-infected hepatocytes [7].

Whether both Gn and Gc were essential to form the VLPs was further investigated by expressing only Gc protein, together with N protein. In this construct, we used the same strategy as above except that an extra base was introduced in to the M sequence to create a frame shift in the Gn sequence. As a result, the translation of Gn was terminated after 47 amino acids. After 3 days of infection with the recombinant baculovirus expressing Gc and N, the supernatant was collected. After clarification, the supernatant was further purified by potassium tartrate-glycerol gradient and a visible band was collected. When the purified material from gradient was analyzed by EM a significant number of VLPs with spiky structures on the surface were identified (Fig. 5). These particles exhibited different morphology than those formed by either N protein alone or the VLPs with all three proteins. These Gc/N particles had spiky structure on the surface typical of a membrane glycoprotein but were much more pleomorphic than the VLPs consisted of both glycoproteins (Fig. 5).

The results suggest that Gc was expressed and together with N protein produced spiky virus-like structures. Therefore, it can be hypothesized that Gc (and probably Gn) interact with RNP complex independent of each other during virus infection.

The cell lines used in this study were Spodoptera frugiperda Sf9 and Sf21. Sf9 cells were grown in Sf900II serum-free media (Gibco) and Sf21 cells were growth in TC100 media (Sigma) supplemented with 10% fetal calf serum (FCS). Both cell lines were incubated at 28°C. Recombinant baculoviruses based on Autographa californica nuclear polyhedrosis virus (AcNPV) were propagated in Sf21 cells.

Purified RVFV viral RNAs were obtained from Dr. Mark Outlaw, National Collection for Pathogenic Viruses, Porton Down, UK. Monoclonal antibodies, against Gn, Gc and N were generously provided by Dr. Connie Schmaljohn (USAMRIID, Frederick, MD). Monoclonal antibodies anti-N, anti-G1, anti-G2, and polyclonal antibody against RVFV virus strain Zinga were provided by Dr. Michele Bouloy (Institut Pasteur, Paris, France). For cell surface expression assay anti mouse-fluorescein isothiocyanate (FITC)-conjugated and anti mouse-tetramethylrhodamine isothiocyanate (TRITC)-conjugated (Sigma) were used. For fusion assay purified anti-baculovirus envelope gp64 protein (e-Bioscience) was used.

The full-length cDNA of the M segment was obtained by reverse PCR using primers 5'-ACGCGTGTCGACACACAAAGATGGTGCATTAAATGTATG-3' and 5'-GAATTCAGATCTACACAAAGACCGGTGCAACTTC-3', and the cDNA of the N protein coding region was generated by reverse PCR using primers 5'-GTCGACGGATCCCCATGGACAACTATCAAGAGCTTCG-3' and 5'-CTCGAGGAATTCAGATCTTAGGCTGCTGTCTTGTAAGCC-3'. The PCR products were cloned into pM83B [42] and translation context sequences were added by site-directed mutagenesis before the 4th ATG for the Gn/Gc with primer 5'-GGTCTTCCATGGCGGCCGCCCGGGCTG CATCCAAC-3', or before the start codon of the N protein with primer 5'-GTTGTCCATGGCGGCCGCGTCGACCTGCAG-3'. The fragment containing the N ORF and the context was transferred to the transfer vector pRN16 (generated in Roy's lab, unpublished), derived from CL29 [43], to produce pRN-N. The fragment including the context and the sequence from the fourth ATG to the end of the Gn was inserted to pRN16 to obtain pRN-4th Gn/Gc. The EcoRV-KpnI fragment of pRN-4th Gn/Gc, which contained the polyhedron promoter and the Gn/Gc genome, was inserted to pRN-N to construct pRN-Ns-4th Gn/Gc. A sequence containing an extra base, C, between the 625^th and 626^th nucleotides of the M segment was inserted into pRN16 to create pRN-Ns-Gnmut/Gc. This mutation introduces a frame shift after translating 47 amino acid of the Gn and stopped after 8 additional amino acids.

Bacmid BAc10:KO₁₆₂₉ [44] DNA was cotransfected with transfer vectors pRN-N, pRN-N-4^thGn_mut/Gc or pRN-N-4^thGn/Gc into Sf21, to obtain recombinant baculoviruses containing respective expression cassettes. A modified protocol was used to combine the cotransfection and plaque assay, and individual plaques were picked after six days. The recombinant baculoviruses were amplified in Sf21 cells and virus stocks were stored at 4°C. Insect cells were infected with the recombinant virus stocks to examine the recombinant protein expression and VLP production.

Protein expression was analyzed by SDS-polyacrylamide (7.5 to 10%) gels (PAGE) [45]. Proteins were either stained with Commassie brilliant blue or transferred to a cellulose nitrate membrane (Schleicher & Schuell) using a semi-dry transfer cell (Bio-Rad) for Western blotting [46]. Monoclonal antibodies against RVFV Gn, Gc or N proteins diluted 1:1000 in 2% (w/v) milk-phosphate buffer (PBS) were incubated with membranes for one hour. The secondary antibody (anti-mouse IgG conjugated with alkaline phosphatase) (Sigma) was diluted 1:10000. The membranes were finally developed with BCIP-NBT substrate (Sigma).

For VLP purification Sf9 cells were infected with a recombinant baculovirus expressing N and either Gn/Gc polyprotein or Gc for 4 days. Infected cell medium was harvested and after clarification and ultracentrifugation as before, the pellet was resuspended in 20 mM Tris-HCl pH 8.0 and layered on top of step sucrose gradient 20%, 30% and 40% (w/v) [47] and centrifuged for 4 hours at 190000 × g at 4°C. Alternatively, the sample was purified through a potassium tartrate-glycerol gradient [48] by centrifugation for 18 hours in SW 28 rotor at 28,000 rpm. Visual band or fractions of 0.5 ml were collected and analyzed for the presence of RVFV proteins. Positive fractions were diluted with TNE buffer and ultracentrifuged through a sucrose cushion. The pellet was resuspended in TNE buffer and stored at 4°C.

Sf9 cells were grown in monolayers and then infected with the recombinant baculovirus expressing Gn, Gc and N at MOI of 0.5. At 24 hours post-infection an antibody against baculovirus gp64 was added to the media. After 2 hours the media was replaced with low pH media and incubated further for 2 minutes and then replaced with normal pH medium. Incubation was continued approximately 2 hours until syncytia were visible. As a control, Sf9 cells were infected at MOI of 0.5 with a baculovirus expressing Bluetongue virus (BTV) VP2 protein. Syncytia were counted by visual microscopy at 100× magnification.

A purified sample was spun in a micro-centrifuge at full speed for 10 minutes. An aliquot of the supernatant was placed onto a carbon-coated grid, dried with the edge of a piece of filter paper and stained with a drop of 3% phosphotungstic acid (PTA) pH 6.8 [7]. All samples were examined using a Jeo 1200 EX transmission microscope.

Cultured Sf9 cells were collected by spinning down at 1000 rpm for 2 minutes and washed once with serum-free fresh culture medium. The final cell pellet was fixed with 2% glutaraldehyde in serum-free fresh culture medium and embedded in agar, and cut into smaller cubes. The cubes were embedded in epoxy resin and ultra sections were cut, mounted onto formva-coated grid, and stained with 2% uracil acetate, pH 5.5 [7].