Expression of mutated forms of the bovine coronavirus hemagglutinin-esterase protein in stably transformed mammalian cells

The hemagglutinin-esterase protein Is one of three integral membrane glycoproteins on the surface envelope of the hemagglutinating coronaviruses. It is found as a140 kD homodimer composed of disulfide linked monomers of 65 kD. This glycoprotein was first identified and characterized in our laboratory during studies on the bovine coronavirus. The hemagglutinin-esterase gene was recently cloned from the bovine coronavirus RNA genome, sequenced, and transiently expressed in vitro in our laboratory.

To develop an in vitro system to study the synthesis and expression of viral glycoproteins, the hemagglutinin-esterase gene was subcloned into an expression vector, transfected into recipient mammalian cells, and cell lines containing the gene were established. Stably transfected cells were shown to contain the integrated hemagglutinin-esterase gene as determined by polymerase chain reaction amplification of specific gene sequences. Polymerase chain reaction products were shown to be viral specific by Southern blots probed with hemagglutinin-esterase gene specific oligonucleotides.

Despite stable transfection and propagation of clonally selected cell lines, the hemagglutinin-esterase protein could not be detected as a 140 kDA dimer that would reduce to a 65 kD monomer. Abnormal products of the hemagglutinin esterase gene were detected by immunoprecipitation with hemagglutinin esterase specific polyclonal and monoclonal antiserums. These suspected mutated forms of the hemagglutinin-esterase protein were approximately ICQ kD and 60 kD in size and did not reduce to smaller species. Polyclonal antiserum directed against the hemagglutinin-esterase protein (gp65) detected low levels of hemagglutinin-esterase protein in the cytoplasm of transfected cells by indirect immunofluorescence. Transfected cells expressing these mutated forms did not exhibit hemadsorption.

Hypotheses explaining how mutated forms of the HE protein might have arisen during stable transformation are put forth: (1) Necessary viral gene sequences required for the correct transcription of the hemagglutinin-esterase gene may have been deleted during integration of the plasmid DNA Into the cell genome during replication; (2) Expression of the complete hemmagglutlnlnesterase gene may have been toxic to the host cell resulting In the selection of only mutated forms of the gene.

One striking observation was that uninfected human rectal tumor cells, one of the host cells used In this expression system, contain a disulfide-linked dimeric protein of 200kD (reducible to subunits of 95 kD) that Is specifically Immunoprecipitated with one of the hemagglutinin-esterase specific monoclonal antibodies. Since this cellular protein and the hemagglutinin-esterase protein are both disulfide-linked dimers, the Intriguing possibility arises that the hemagglutinin-esterase protein and the Immunoprecipitated cellular protein are evolutionarily related. Taken one step further, perhaps this coronavirus surface envelope glycoprotein originated from a cellular protein. Such a striking structural homology between a viral and cellular protein suggests there may be mimicry of a cell surface protein by the hemagglutinin-esterase protein during Infection leading to pathological consequences.