Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli.
Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene. We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus. 1) 14 kDa is a trimer of identical subunits. 2) A monomer binds to itself more strongly than to a dimer or a trimer. 3) Oligomerization does not require cellular factors. 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity. 5) 14-kDa binds with specificity to the cell surface of cultured cells.
Lai
C F
CF
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
Gong
S C
SC
Esteban
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005814:Genes, Viral; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009500:Neutralization Tests; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014616:Vaccinia virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins