Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR.
The structural membrane proteins prM and E of the flavivirus tick-borne encephalitis (TBE) virus were expressed in mammalian cells for the purpose of probing the structure and molecular interactions of these proteins. Advantage was taken of the natural error frequency of the Taq polymerase used in the PCR amplification to generate a randomly mutated population of genes that were then cloned directly into plasmid expression vectors under the control of an SV40 promoter. Analysis of the mutation frequency by direct sequencing of 22 separate clones showed that the PCR produced mutations at a rate yielding an average of one to two amino acid changes per clone in the 496 amino acid long protein E. This is an ideal rate for assessing the importance of individual amino acid residues within protein domains, thus demonstrating the potential value of the PCR as a random mutagenesis method. Clones encoding wild-type prM and E proteins, and a truncated form of E, were also constructed by recombining portions of selected PCR clones. Transfection of COS-1 cells with these constructs resulted in expression of the prM and E proteins, which was demonstrated by indirect immunofluorescence using monoclonal antibodies (Mabs). The intracellular level of TBE virus antigen, measured in lysates of transfected cells by ELISA, reached approximately 25% of that found in virus-infected COS cells. Furthermore, it was shown by immunofluorescence using a panel of 19 anti-E Mabs that the antigenic structure of the expressed E proteins was nearly identical to that of E protein in infected cells, thus confirming the suitability of this model system as a tool for studying flavivirus protein structure.
Institute of Virology, University of Vienna, Austria.
S L
SL
Allison
C W
CW
Mandl
C
C
Kunz
F X
FX
Heinz
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015752:Genes, env / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D014162:Transfection / Q000379:methods; D014759:Viral Envelope Proteins / Q000096:biosynthesis*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus; C065696:glycoprotein E, Flavivirus