Characterization of baculovirus-expressed Rift Valley fever virus glycoproteins synthesized in insect cells.
A cDNA corresponding to the complete coding region of the M RNA of the M12 mutant of Rift Valley fever virus (RVFV) strain ZH548 (K. Takehara, M-K. Min, J.K. Battles, K. Sugiyama, V.C. Emery, J.M. Dalrymple, and D.H.L. Bishop, Virology, 169, 452-457, 1989) has been inserted into the baculovirus transfer vector pAcYM1. By comparison with the parent RVFV, the M RNA of the M12 mutant has a new small open reading frame (ORF1) upstream of the one that initiates the precursor of the viral glycoproteins (ORF2, gene order: NS(M)-G2-G1). A derivative of the M12 cDNA was prepared from which most of the upstream sequences (including a polyT tract and ORF1) were removed. Other cDNA constructs were made from this derivative, constructs in which most of the G1 sequences were also removed, or most of the NS(M) coding sequences, or all of the NS(M) and most of G2 coding sequences. Each RVFV M cDNA construct was inserted into a pAcYM1 transfer vector and recombinant baculoviruses were produced (RVM1-5). The derived viruses were employed to study the expression and properties of the RVFV glycoproteins in Spodoptera frugiperda insect cells. For each recombinant virus evidence was obtained which indicated that the RVFV glycoproteins were produced and processed in the insect cells. Although four of the recombinants gave low expression levels of the RVFV glycoproteins, for the vector that made only the G1 product, the expression level was significantly higher. Immunofluorescence analyses established that the RVFV glycoproteins were present both at intracellular locations and on the surface of the recombinant baculovirus infected insect cells.
Takehara
K
K
NERC Institute of Virology and Environmental Microbiology, Oxford, U.K.
Morikawa
S
S
Bishop
D H
DH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009036:Moths; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012296:Rift Valley fever virus / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D014759:Viral Envelope Proteins