Ligand-Induced Activation of an Engineered Viral Fusion Protein

Ligand-induced Activation of an
Engineered Viral Fusion Protein

Introduction

Influenza
is a pathogenic virus with various human and avian sub-types. In influenza, a
membrane protein called hemagglutinin (HA) sits on the surface of the virus in
an inactive state with its fusion peptide imbedded within the protein. The
fusion peptide is a 20-amino acid sequence that plays a dynamic role in the
infection of target cells. When exposed to a low pH environment, the protein
will irreversibly change its structure so that the fusion peptide relocates and
becomes exposed. HA is a model membrane fusogen and understanding its function
will better inform attempts to design fusion activity into drug or gene
delivery agents. The research objective is to characterize an engineered HA’s
activation in response to ligand binding and to determine if autocatalytic
activation, previously observed for a HA of the H7 subtype, occurs in other HA
subtypes.

Methods

Through
Polymerase Chain Reaction (PCR), a primer encoded with a tetra-Cys motif
replaced a section of the fusion peptide of HA and then was cloned back into
the full sequence. The CCPGCC tag was placed at various starting locations
within the fusion peptide’s 20 amino acid sequence and cell lines of these
mutants were created by transfection of Chinese Hamster Ovary (CHO) cells. By
altering the sequence, it allows FlAsH, which is a fluorescent dye that binds
to the tetra-Cys sequence, to bind to the fusion peptide when it is exposed.
Once FlAsH binds, the dye fluoresces allowing the structural change and
location of the protein to be seen within the cell via fluorescent video
microscopy and to be quantified by flow cytometry.

 Results

A
5-minute period of low pH exposure is necessary for the activation of a few HA
on the cell surface, and, even after the removal of low pH environment, HA
still activates fully on the surface of the cell. It was observed that without
a low pH pulse, HAs were still being induced after a 7-minute incubation period
with FlAsH, suggesting that FlAsH interacts with the fusion peptide upon
binding to induce the conformation change. This was further tested by a
real-time microscopy experiment of FlAsH and a neutral pH environment.

Discussion

The continuing activation of HA
after the removal of an acidic stimulant suggests a disturbance in the membrane
that causes all HA to activate fully across the cell or that some other type of
communication occurs to create a cell-wide activation. The conformational
change being driven by FlAsH provides an interesting avenue for activation of
cell membrane fusion. It will make it possible to activate the conformational
change without dependence on acid sensitivity but rather the presence of a
ligand. These experiments were demonstrated with the avian influenza H7 subtype
of HA. This work is currently being extended to the human-infectious influenza
H3 subtype of HA. The dynamics of this subtype are proposed to be different due
to the structure differences between the subtypes.