High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors.
We have constructed a collection of simian virus 40 (SV40) plasmid vectors useful for transient or constitutive expression of cDNA or genomic DNA in animal cells. Most vectors contain several unique restriction sites downstream from the SV40 late or early promoter, and are available with or without the virus-specific splicing signals. The use of these vectors for transient expression in monkey cells of X47 (H3N2) influenza hemagglutinin (HA) and matrix protein (M1) was demonstrated. Membrane-bound (HAm) as well as secreted forms of the HA glycoprotein lacking the sequence of the C-terminal anchor (HA-) have been obtained. Depending on the insert, the type of vector and the amount of transfected DNA, HA levels in COS cells [Gething and Sambrook, Nature 293 (1981) 620-625] transfected with late replacement SV40 vectors vary from 10(9) (HAm) to 10(8) (HA-) molecules per transfected cell. The maximum expression levels with early replacement vectors in COS cells are at least 50 times lower. In addition to the optimalization and the characterization of the expression of each vector-coded influenza protein, cotransfections, including vectors expressing HAm, neuraminidase (NA) and M1, were undertaken. The latter experiments did not result in a measureable amount of HAm or NA in the cell culture medium, suggesting that expression of these three structural viral proteins does not result in budding of (empty) influenza particles from the cell surface.
Huylebroeck
D
D
Laboratory of Molecular Biology, State University of Gent, Belgium.
Maertens
G
G
Verhoeyen
M
M
Lopez
C
C
Raeymakers
A
A
Jou
W M
WM
Fiers
W
W
D000914:Antibodies, Viral / Q000302:isolation & purification; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006388:Hemagglutinins / Q000032:analysis; D009980:Influenza A virus / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D011233:Precipitin Tests; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics*
D000914:Antibodies, Viral; D006388:Hemagglutinins; D014764:Viral Proteins