Related Experiment Videos
Cell surface expression of biologically active influenza C virus HEF glycoprotein expressed from cDNA
1Howard Hughes Medical Institute, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Insights
The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus mediates key functions independently. Unlike previous findings, HEF-AA and HEF-Tay show abundant cell surface expression without viral co-factors.
Area of Science:
- Virology
- Molecular Biology
- Glycoprotein Function
Background:
- Influenza C virus hemagglutinin, esterase, and fusion (HEF) glycoprotein mediates receptor binding, destruction, and membrane fusion.
- Previous studies suggested a short cytoplasmic tail (Arg-Thr-Lys) in HEF-JHB inhibits cell surface expression.
Purpose of the Study:
- To investigate the cell surface transport and functional activities of HEF glycoproteins from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses.
- To determine if the cytoplasmic tail sequence affects HEF cell surface expression and function.
Main Methods:
- Cloning and expression of HEF cDNAs from HEF-AA and HEF-Tay.
- Monitoring HEF transport using trypsin cleavage, indirect immunofluorescence microscopy, and flow cytometry.
- Assessing HEF binding to human erythrocytes, membrane fusion activity, and esterase activity.
Main Results:
- HEF-AA and HEF-Tay showed abundant cell surface transport, independent of other viral proteins.
- Their cell surface expression was not inhibited by the cytoplasmic tail sequence, contrary to previous findings with HEF-JHB.
- HEF-AA and HEF-Tay demonstrated receptor binding, low-pH/trypsin-dependent membrane fusion, and esterase activity.
Conclusions:
- The cytoplasmic tail sequence does not inhibit cell surface expression of HEF-AA and HEF-Tay.
- HEF glycoproteins from these strains are efficiently transported to the cell surface without co-expression of other viral proteins.
- The HEF glycoprotein alone mediates receptor binding, membrane fusion, and esterase activities, demonstrating its multifunctional role.
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.