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


CONCLUSIONS
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.
Liu
Li
L
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. li.liu@qmul.ac.uk
Celma
Cristina C P
CC
Roy
Polly
P
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012296:Rift Valley fever virus / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D014773:Virology / Q000379:methods
D006023:Glycoproteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins