Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cDNAs in prokaryotic and eukaryotic cells

To study the biological and immunological properties of influenza virus surface glycoproteins, cDNA copies of the haemagglutinin (HA) and the neuraminidase (NA) genes of A/WSN/33 influenza virus were cloned and expressed in prokaryotic and eukaryotic cells. In Escherichia coli, maximum expression of HA is obtained only as a fusion protein in which the NH₂-terminal portion is provided by a bacterial protein (i.e. βgal or trpLE′). The HA expressed in bacteria (bacterial HA) is recognized by polyclonal anti-WSN antibodies but not by neutralizing monoclonal antibodies. The antibodies made against the bacterial HA bind to the detergent-treated viral HA, intact virus and live influenza infected cells, but fail to show either haemagglutination inhibition (HI) or virus neutralization. These results suggest that the three-dimensional structure as well as the antigenic epitopes of the bacterial HA are different from that of native viral HA. HA, expressed from cDNA in cultured animal cells, is shown to possess the structural features of the native viral HA. It is glycosylated, transported to the apical domain of the plasma membrane of polarized cells, causes haemadsorption and can induce cell to cell fusion at low pH after proteolytic cleavage. An attempt was made to define the structural features of HA required for sorting and directional transport by making chimeras with vesicular stomatitis virus G (VSV G) protein either by switching the amino terminus or the carboxy terminus of HA with that of VSV G. These chimeric proteins were translocated across the rough endoplasmic reticulum (RER) but were blocked in transport between the RER and cell membrane. These preliminary results indicate that the three-dimensional structure, in addition to specific sequences, may play a critical role in the transport process. More precise constructions are in progress to delineate the functions of the different domains of HA molecule. HA has been expressed in the lower eukaryote Saccharomyces cerevisiae (baker's yeast). The HA expressed in yeast (yeast-HA) is glycosylated and membrane-bound. It is recognized by both the polyclonal and neutralizing monoclonal antibodies made against the native HA. These results suggest that the yeast-HA retains the antigenic epitopes present in the native viral HA and therefore may be of significance in the development of a subunit vaccine. NA, the other influenza glycoprotein, has been expressed from the cloned cDNA in cultured monkey kidney cells and the expressed NA, like the native viral NA, is glycosylated and enzymatically active. Furthermore, it has been shown that the NH₂-terminus of NA, in addition to providing the anchor function, also provides the signal function for translocation across the rough endoplasmic reticulum (RER). Like HA, NA expressed from cloned cDNA is preferentially transported to the apical domain of the plasma membrane of polarized epithelial cells.