Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins - PubMed (original) (raw)
Rift Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins
Li Liu et al. Virol J. 2008.
Abstract
Background: Studies on Rift Valley Fever Virus (RVFV) infection process and morphogenesis have been hampered due to the biosafety conditions required to handle this virus, making alternative systems such as recombinant virus-like particles, that may facilitate understanding of these processes are highly desirable. In this report we present the expression and characterization of RVFV structural proteins N, Gn and Gc and demonstrate the efficient generation of RVFV virus-like particles (VLPs) using a baculovirus expression system.
Results: A recombinant baculovirus, expressing nucleocapsid (N) protein of RVFV at high level under the control of the polyhedrin promoter was generated. Gel filtration analysis indicated that expressed N protein could form complex multimers. Further, N protein complex when visualized by electron microscopy (EM) exhibited particulate, nucleocapsid like-particles (NLPs). Subsequently, a single recombinant virus was generated that expressed the RVFV glycoproteins (Gn/Gc) together with the N protein using a dual baculovirus vector. Both the Gn and Gc glycoproteins were detected not only in the cytoplasm but also on the cell surface of infected cells. Moreover, expression of the Gn/Gc in insect cells was able to induce cell-cell fusion after a low pH shift indicating the retention of their functional characteristics. In addition, assembly of these three structural proteins into VLPs was identified by purification of cells' supernatant through potassium tartrate-glycerol gradient centrifugation followed by EM analysis. The purified particles exhibited enveloped structures that were similar to the structures of the wild-type RVFV virion particle. In parallel, a second recombinant virus was constructed that expressed only Gc protein together with N protein. This dual recombinant virus also generated VLPs with clear spiky structures, but appeared to be more pleomorphic than the VLPs with both glycoproteins, suggesting that Gc and probably also Gn interacts with N protein complex independent of each other.
Conclusion: Our results suggest that baculovirus expression system has enormous potential to produce large amount of VLPs that may be used both for fundamental and applied research of RVFV.
Figures
Figure 1
Expression and purification of RVFV nucleoprotein (N) protein. Insect _Sf_9 cells were infected with a recombinant baculovirus expressing RVFV N protein and four days after infection the expression of N was assessed. A) Infected cell lysate expressing N protein was analyzed by SDS-PAGE followed by Commassie Brilliant blue staining (lane 2) or Western blotting (lane 4) and compared with total proteins from uninfected insect cells (lanes 1 & 3). Protein markers were included and sizes in kilo-Dalton (kDa) are shown at the right. B) Purification of N protein by gel filtration. The position of the peak correspondent to N and the relative elution position of molecular markers are indicated. C) Samples of the gel filtration fractions corresponding to the peak of protein were analyzed by SDS-PAGE and stained with Commassie brilliant blue (lanes 2 to 9). An aliquot of a fraction, prior to N protein fraction, was included as a control (lane1). The relative position of molecular marker is indicated in KDa. D) Purified N protein was analyzed by SDS-PAGE followed by Commassie Brilliant blue staining (lane 2) or Western blotting (lane 3) and compared with total proteins from infected insect cells (lane 1). Protein markers were included (lane M) and sizes in KDa are shown at the right. E) An aliquot of purified N protein were negatively stained with 3% phosphotungstic acid (PTA), pH 6.8 and visualized by electron microscopy. A particulate structure is indicated with an arrow in upper panel and lower panel shows amplified particles. Bar represents 100 nm.
Figure 2
Detection of RVFV Gn and Gc and the cell surface expression. A) Cell lysate from infected cells with a recombinant baculovirus expressing RVFV N, Gn and Gc were analyzed by SDS-PAGE followed by Commassie blue stain (lane2). As a control cell lysate from uninfected cells were included (lane 1). B) Western blot using specific antibodies against Gn (lane 2) or Gc (lane 5) was performed with cell lysate expressing RVFV proteins N, Gn and Gc. As a control cell lysates from uninfected cells (lanes 1 and 4) or expressing RVFV N protein (lanes 3 and 6) were included. C) Cell surface expression of RVFV Gn. Infected cells expressing RVFV N, Gn and Gc proteins were fixed and processed for immunoflorescence under non-permeabilizing conditions. To detect RVFV Gn protein, a specific antibody was used followed by an anti-mouse-FITC conjugated secondary antibody (upper panel). As control cells expressing β-Gal protein were processed similarly (lower panel). D) Cell surface expression of RVFV Gc. Cells expressing RVFV N, Gn and Gc were examined for cell surface expression of Gc using a specific antibody against Gc and a anti mouse-TRITC as secondary antibody (upper panel). Control cells were included (lower panel).
Figure 3
Fusogenic activity of RVFV Gn and Gc proteins. A) Insect cells were infected with a baculovirus expressing RVFV N, Gn and Gc for 24 hours and a monoclonal antibody against baculovirus gp64 were added to the media. After 2 hours the media was replaced with low pH media (pH 5.0) for two minutes and then replaced with normal media (right, upper panel). As controls, infected cells expressing RVFV proteins were kept at normal pH media of 6.5 (left, upper panel). As negative control, infected insect cells expressing BTV VP2 protein were included and pH shift was performed (right, lower panel) or the media was kept at neutral pH (left, lower panel). Pictures were taken at 200× magnification. B) Quantification of fusion capacity. The number of syncytia per field was counted by visual microscopy at 400× magnification and the average and standard deviation were calculated. Each assay was performed in triplicate.
Figure 4
Expression of N, Gn and Gc proteins produced virus-like particles. A) _Sf_9 cells were infected with the recombinant baculovirus expressing RVFV N, Gn/Gc proteins and after 4 days both infected cells and the media were harvested. An aliquot from the infected cell lysate was analyzed by SDS-PAGE and stained by Commassie Brilliant blue (lane1). The media was clarified followed by ultracentrifugation on a potassium tartrate-glycerol gradient. An aliquot of purified material was analyzed as before (lane 2). Confirmation of viral proteins in purified samples was performed by Western blotting using monoclonal antibodies against either Gn (lane3) or N (lane 4) or with a polyclonal antibody against RVFV Zinga strain (lane 5). Protein markers were included (lane M) and sizes in kDa are shown on the right. B) Negative staining of purified VLPs. The spiky structures of the particle surface units consisting of glycoproteins Gn and Gc are indicated by arrows (upper panel). The spiky surface units are indicated by arrows (lower panels). The number of the surface unit of each particle is indicated at the upper left corner. Bar represents 100 nm. C) EM of infected cells' section showing VLPs are released into vacuoles. Note the presence of particles (black arrow) within the membrane (white arrow) of the vacuole boundaries. D) Same showing virus inclusion body in the cytoplasm indicated by arrows.
Figure 5
Assembly of VLPs by expression of RVFV N and Gc proteins. The supernatant of cells expressing N and Gc was purified as described in Methods and a sample of the purified material was stained and analyzed by EM. VLP structures with variable shapes and sizes were detected.
References
- Peters CJ, Liu CT, Anderson GW, Jr., Morrill JC, Jahrling PB. Pathogenesis of viral hemorrhagic fevers: Rift Valley fever and Lassa fever contrasted. Rev Infect Dis. 1989;11 Suppl 4:S743–9. - PubMed
- Centers for Disease Control and Prevention Outbreak of Rift Valley fever - Saudi Arabia, August - October 2000. Morb. Mortal. Wkly. Rep. 2000. pp. 905–908. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous