Scientists at the University of Maine (UMaine) and National Institutes of Health have identified a putative interaction between a key influenza A virus protein and a lipid in the cell surface membranes of the host cells that it infects, which if verified could provide pointers to the development of new antiviral therapies.

The team’s studies, using confocal and super-resolution microscopy to investigate the spatial patterning of the viral hemagglutinin (HA) protein and host cell phosphatidylinositol 4,5-bisphosphate (PIP2) lipid in living cells in vitro, showed that the two molecules were tightly colocalized in cell plasma membranes (PMs). “Our findings show for the first time a connection between the influenza virus surface protein HA (the H in H1N1) and the host cell lipid PIP2,” commented UMaine professor of physics Samuel Hess, PhD, who headed the research. “With further single-molecule microscopy experiments, we are now testing the hypothesis that a certain region within HA could be the site of interaction with PIP2.” Hess developed the patented super-resolution microscopy technique used to image the cells and follow the spatial clustering and patterning of the host and viral molecules in the cell membranes.

The team reported on its findings in the Biophysical Journal, in a paper titled, “Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering.”

Influenza viruses belong to the family Orthomyxoviridae. The virus particles, which the NCBI states are about 80–120 nm in diameter and can be spherical or pleomorphic in shape, are bounded by a lipid membrane envelope that is studded with two glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA has two roles, according to the Centers for Disease Control and Prevention. The protein allows the flu virus to enter healthy cells, and it also acts as one of the antigens that triggers the host’s immune response to protect against reinfection by the same strain. However, mutations in HA and to a lesser extent NA allow the virus to change its antigens and so sidestep recognition by the immune system. Most seasonal flu vaccines are designed to target the HA of those flu virus strains that research indicates will be most prevalent during that flu season.

The lipid envelope of the flu virus is generated from host cell membranes, and during this process, the viral HA and NA glycoproteins are inserted into the viral envelope, the authors continued. “HA localized to the PM of host cells clusters spontaneously and is crucial for fusion, viral budding, and infection.”

PIP2 in cell plasma membranes controls cellular functions by modulating signaling pathways. Many of these pathways control the actin cytoskeleton, which acts as a structural framework for cell shape, motility, and membrane organization. During flu infection, viral manipulation of signaling pathways allows it to suppress innate immune responses, keep infected cells alive, and increase the rate at which new viral particles can be constructed.

PIP2 forms nanoscopic clusters in cell plasma membranes, but the processes that underpin PIP2 mobility and its spatial patterning are not well understood, the authors further commented. “Although the lateral organization of proteins and lipids (clustering) in the cell plasma membrane (PM) is crucial to diverse fundamental cellular processes, there is considerable disagreement on the organizational mechanisms that govern such clustering …”

Many proteins that have been seen together with HA are known to control the actin cytoskeleton, and they also have known binding to PIP2, but the connection hasn’t previously been explained. In their reported studies, the researchers used the confocal and super-resolution microscopy techniques to image HA and PIP2 in different living cell types. As well as observing that the two molecules commonly colocalized to the same regions of the plasma membrane, the team also discovered that HA and PIP2 affected each other’s motions. The presence of HA was linked with PIP2 moving more slowly, reversing direction more frequently, and forming more highly confined clusters.

When PIP2 was present the density of HA also increased. It is this high density of HA on the viral surface that is required to allow viral entry into host cells by membrane fusion. “Taken together with the observation that PIP2 clusters depend on local HA density, these results suggest an interdependence of PIP2 and HA cluster properties on one another,” the authors concluded. “Our observations also show that PIP2 clustering is significantly altered in the presence of HA, HA can modulate the mobility of PIP2, the measured spatial profile of HA clusters predicts the behavior of PIP2, and the measured distributions of HA and PIP2 in clusters parallel each other on average.”

Hess and colleagues are now testing whether a region within the HA’s cytoplasmic tail region could be interacting with PIP2. The HA tail is relatively conserved, which indicates that it could represent a potential drug target. Their results also suggested that the mechanism of any interaction between HA and PIP2 may differ from other known mechanisms.

“In contrast to other mechanisms of protein-lipid interactions such as ordering of molecules into lipid rafts, lipid confinement by protein fences, tethering of lipid motion, or buffering by fixed binding sites, our findings describe and explain spatial PIP2 distributions and how they change in time via a distinctly dynamic mechanism—a potential gradient due to binding sites that are themselves both mobile and clustered,” they wrote. “… This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.”

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