Expression and secretion of Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus.
The nonstructural protein NS1 of Japanese encephalitis virus (JEV) was expressed at a high level under the control of the polyhedrin promoter in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus. Recombinant NS1 was designed to contain its natural signal sequence at its N-terminus and no C-terminal hydrophobic domain that could act as a membrane anchor. This recombinant protein exhibited similar size to native NS1 expressed in Aedes albopictus (C6/36) insect cells infected with wild-type JEV. The signal sequence of NS1 allowed translocation of the protein into the endoplasmic reticulum where it underwent glycosylation. A small fraction of synthesized NS1 was able, in the absence of any other viral protein, to associate as a homodimer, showing similar characteristics to the native dimer. Interestingly, this recombinant dimeric form seemed to be exported and released in the extracellular medium of infected cell culture. During its transport, one of the two N-linked oligosaccharides of the polymannose type was processed to an endoglycosidase H-resistant form, suggesting that the protein had passed through the Golgi compartment before reaching the cell surface. Moreover, Triton X-114 partitioning analysis showed that monomeric NS1 behaved essentially as a hydrophilic protein, whereas both intracellular and extracellular dimeric NS1 were either free of or associated to membraneous components.
Flamand
M
M
Laboratoire des Arbovirus, Institut Pasteur, Paris, France.
Deubel
V
V
Girard
M
M
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001692:Biological Transport; D002478:Cells, Cultured; D004664:Encephalitis Virus, Japanese / Q000235:genetics*; D006031:Glycosylation; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009036:Moths; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins