Production of Influenza C HEF pseudotyped lentiviral particles

Abstract

While influenza A and B virus contain the two glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) inserted into the viral membrane, influenza C virus possesses only one spike designated Hemagglutinin-Esterase-Fusion (HEF) protein which combines the functions of both HA and NA. Like HA, it recognizes and binds to a receptor on the cell surface to initiate virus entry. However, the receptor is N-acetyl-9-O-acetylneuraminic acid. HEF is the receptor-destroying enzyme, which is the function of the neuraminidase (NA) in influenza A and B virus. Although Influenza C virus infections are generally mild and self-limited, it comprises around 13 % of influenza positive cases in hospitalized children. It is also a cause of community acquired pneumonia of children. As influenza research has been more focused on Influenza A and B, it is of interest to pseudotype influenza C HEF. The full sequence of Inflenza C HEF (C/Tokyo/4/2014) was obtained and cloned into pi.18 expression plasmid. The second generation packaging construct pCMVΔR8.91, and the self-inactivating lentiviral vector pCSFLW expressing firefly luciferase, and pCSGW expressing GFP were used for lentiviral vector particle production. The original HEF sequence was mutated by site directed mutagenesis to inhibit esterase activity to prevent receptor destruction prior to pseudotype viral entry. Pseudotyped particles bearing the HEF glycoprotein were successfully produced and verified by fluorescence and Luciferase titration assay with titres of 5×107 RLU/ml. Currently, optimization is undergoing to achieve higher titres as well determining the specific protease/s utilized by HEF for its activation.