Cell Surface Expression of Biologically Active Influenza C Virus HEF Glycoprotein Expressed from cDNA

Abstract

The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus possesses receptor binding, receptor destroying, and membrane fusion activities. The HEF cDNAs from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses were cloned and expressed, and transport of HEF to the cell surface was monitored by susceptibility to cleavage by exogenous trypsin, indirect immunofluorescence microscopy, and flow cytometry. Previously it has been found in studies with the C/Johannesburg/1/66 strain of influenza C virus (HEF-JHB) that transport of HEF to the cell surface is severely inhibited, and it is thought that the short cytoplasmic tail, Arg-Thr-Lys, is involved in blocking HEF cell surface expression (F. Oeffner, H.-D. Klenk, and G. Herrler, J. Gen. Virol. 80:363–369, 1999). As the cytoplasmic tail amino acid sequences of HEF-AA and HEF-Tay are identical to that of HEF-JHB, the data indicate that cell surface expression of HEF-AA and HEF-Tay is not inhibited by this amino acid sequence. Furthermore, the abundant cell surface transport of HEF-AA and HEF-Tay indicates that their cell surface expression does not require coexpression of another viral protein. The HEF-AA and HEF-Tay HEF glycoproteins bound human erythrocytes, promoted membrane fusion in a low-pH and trypsin-dependent manner, and displayed esterase activity, indicating that the HEF glycoprotein alone mediates all three known functions at the cell surface.

Epidemiology and Clinical Characteristics of Influenza C Virus

“…Influenza A and B surface proteins include hemagglutinin (HA) and neuraminidase (NA), which mediate attachment, entry, and escape [25,31]. In contrast to influenza A and B, ICV hemagglutinin-esterase-fusion (HEF) glycoprotein, encoded on segment 4, efficiently fulfills the roles of both HA and NA by facilitating host receptor binding, cleaving sialic acid, and membrane fusion [32][33][34][35]. However, ICV HEF binds to N-acetyl-9-O-acetylneuraminic acid rather than to N-acetyl-neuraminic acid for influenza A and B viruses [36].…”

Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses

“…Virions of influenza A and B viruses are studded with two different types of spikes, the hemagglutinin (HA), which mediates receptor-binding and pH-dependent membrane-fusion and the neuraminidase (NA), the viral RDE [20]. In contrast, influenza C virus possesses only one type of spike, which combines all three functions [21][22][23][24][25][26][27] and hence is commonly referred to as the hemagglutinin-esterase-fusion protein (HEF). Like its homologue HA, to which it bears limited yet significant primary sequence similarity [28][29][30][31], HEF is a homotrimer of an N-glycosylated type I membrane protein [32][33][34].…”

The role of stearate attachment to the hemagglutinin-esterase-fusion glycoprotein HEF of influenza C virus

“…2A). Two bands representing the HEF1 subunits became visible in the fluorogram only after 90 min of labelling confirming that intracellular transport of HEF is slow in comparison to HA (Pekosz et al, 1999, Szepanski et al, 1994. The amount of HEF1 greatly increases with longer labelling time, whereas the band representing the HEF0 precursor remains (almost) constant.…”

“…First we compared transport of wt and non-acylated HEF to the plasma membrane by cell surface trypsinization. The enzyme cleaves HEF into its subunits HEF1 and HEF 2, but only if HEF is exposed at the cell surface (Pekosz and Lamb, 1999). Virus-infected MDCKI cells were metabolically S-methionine for 1, 30, 60, 90, 120 and 180 min respectively.…”