“Tagging Influenza Viral Cell Entry Protein-Neutralizing Antibody HC19 for Fluorescent Visualization”
Influenza is an enveloped virus that infects millions of humans each year. Hemagglutinin (HA) is a glycoprotein trimer on the viral surface of influenza. One viral particle will employ hundreds of HAs to bind target cell membrane receptors terminating in residues of a nine-carbon sugar called sialic acid. Following cell attachment, the viral particle is internalized by invagination of the plasma membrane. The virus is thus enclosed within an intracellular membrane compartment called an endosome. A low pH environment within the endosome initiates the conformational rearrangement of HA trimers that results in fusion of the viral and endosomal membranes. Three to five neighboring HAs at the virus-target membrane interface cooperatively participate in viral fusion. A small disturbance in the density of active HAs may delay fusion and reduce viral infectivity. The human immune system can combat influenza virus using anti-HA antibodies, which are typically of the immunoglobulin G (IgG) antibody class. HC19 is an IgG-type antibody that binds to the sialic acid binding domain of HA and prevents viral attachment to the cell.
A concentration at which HC19 cannot prevent viral attachment but is internalized with the viral particle and can impact HA’s ability to mediate membrane fusion remains unexplored. HC19 can be tagged with a peptide recognition motif for an enzyme that attaches a fluorescent molecule to the antibody. Under total internal reflection fluorescence (TIRF) microscopy, the labeled antibody can be observed. Fluorescent visualization of HC19 will allow us to quantify the number of antibodies bound to each viral particle undergoing fusion. From this number, we can record the effects of HC19, at low concentrations, on influenza cell attachment efficiency and membrane fusion kinetics at the virus-endosome interface. To help label the antibody, I constructed an expression vector containing a sequence for peptide-tagged HC19.
Preliminary results show a small-scale purification of the tagged antibody. From this line of research, we can also study the evolving nature of HA and its ability to counteract immune pressure.