Maturation of the dengue-2 virus NS1 protein in insect cells: effects of downstream NS2A sequences on baculovirus-expressed gene constructs.
A series of recombinant baculoviruses was constructed in order to study the influence of downstream NS2A sequences on the processing of the dengue virus NS1 glycoprotein in insect cells. NS1 alone was expressed at a high level in its native dimeric form and processed efficiently through the Spodoptera frugiperda (Sf) cell secretory pathway. Recombinant NS1 was found associated with the plasma membrane and was also secreted into the extracellular medium. Although both intra- and extracellular NS1 were processed to an endo H-resistant form in Sf cells, Triton X-114 phase separation analysis further suggested that some modifications in addition to dimerization account for the hydrophobic properties of NS1, and that N-glycosylation was therefore not the only difference between the cell-associated and secreted forms. Cleavage at the NS1-NS2A junction of these recombinants demonstrated that as few as 26 amino acids from the N terminus of NS2A provide a sufficient, but not optimal, recognition sequence for a functional cleavage mediated by a protease present in Sf cells infected with recombinant Autographa californica nuclear polyhedrosis virus expressing NS1.
Leblois
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Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia. cprehaud@mendel.sis.pasteur.fr
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D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003902:Detergents; C079340:NS1 protein, Dengue virus type 2; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010450:Endopeptidases