Biology subjects

Mannsverk, S.

Publications and source records attributed to Mannsverk, S..

4 recordsLinked to original sources

bioRxiv2026

Tuning nanoscale lipid clustering controls influenza viral binding

Influenza virus attaches to cells by binding to sialylated glycans. Subsequent endocytosis of the virus and endosomal maturation leads to a conformational change in the hemagglutinin coat protein that ultimately results in membrane fusion and release of the viral genome into the host cytoplasm, where it is trafficked to the nucleus and commences replication. Individual hemagglutinin-glycan binding affinities are weak (Kd approximately mM), so multivalency is key to viral attachment. Receptor density or clustering has previously been shown to affect influenza binding, but the overall spatial heterogeneity of both plasma and endosomal membranes has not been fully addressed. Here, we show that nanoscale lipid clustering can increase local concentration of glycosphingolipids and greatly enhance influenza viral binding in a way that macroscopic phase separation does not, and that binding avidity is dependent on the spatiotemporal scales of this clustering. This clustering is driven by sphingolipids and cholesterol and the nanoscale lipid environment of these clusters persists to affect the viral entry process. This finding demonstrates how fine receptor patterning can drive infection and illustrates the importance of tuning spatial scale in liquid-liquid phase separation.

biophysics↗

bioRxiv2025

Influenza viral infection at the plasma membrane is restricted by lipid composition

Many enveloped viruses enter cells via fusion with the endosomal membrane, raising the question whether entry through the endosomal route confers a fitness advantage over fusion directly at the plasma membrane. We found that influenza A virus fusion at the plasma membrane of A549 cells resulted in a 7.6-fold reduction in productive cell infection, compared to infection through the physiological endosomal route. We hypothesized that this was partially explained by restrictive and permissive membrane factors at the plasma and endosomal membrane, respectively. To test this, we developed a single-viral content mixing assay with plasma membrane vesicles (PMVs), where a fluorescent content marker was loaded into purified PMVs at a quenched concentration, through freeze-thawing. We show that influenza fusion with the plasma membrane is 6-fold less efficient than with liposomes containing the endosome-enriched phospholipid BMP. Incorporating BMP into the PMVs with pre-loaded methyl--cyclodextrin (MCD) did not rescue full fusion, suggesting that the lack of BMP in the plasma membrane is not sufficient to explain decreased fusion with the plasma membrane. Depletion of cholesterol from PMVs enhanced lipid mixing rates and reduced membrane order. Lastly, modest depletion of cholesterol from A549 lung epithelial cells rescued influenza infection through fusion at the plasma membrane by 2.4-fold. We propose that for the endosome-adapted influenza virus, fusion directly at the plasma membrane is restricted by the liquid-ordered-like nature of the plasma membrane, raising the question whether the plasma membrane is broadly less permissive to viral entry than endosomes. ImportanceInfluenza virus normally enters cells via endosomes, yet it has been known for decades that acidic conditions can force it to enter via the plasma membrane. Here, we show that the plasma membrane route yields 9-fold worse infection of cells and, surprisingly, most of this effect can be attributed to the plasma membrane being less permissive to viral membrane fusion. This difference between plasma membrane and endosomes cannot be simply abolished by adding a key endosomal lipid that can boost fusion in synthetic membranes. The outer surface of the plasma membrane is known to be highly ordered, and disrupting this order does increase fusion as well as boost the ability of influenza virus to infect cells. We therefore conclude that the complex and ordered composition of cell membranes helps control when and where influenza virus can enter and infect.

biophysics↗

bioRxiv2022

Influenza virus membrane fusion is promoted by the endosome-resident phospholipid bis(monoacylglycero)phosphate

The phospholipid bis(monoacylglycero)phosphate (BMP) is enriched in late endosomal and endo-lysosomal membranes and is believed to be involved in membrane deformation and generation of intralumenal vesicles within late endosomes. Previous studies have demonstrated that BMP promotes membrane fusion of several enveloped viruses, but a limited effect has been found on influenza virus. Here, we report the use of single-virus fusion assays to dissect BMPs effect on influenza virus fusion in greater depth. In agreement with prior reports, we found that hemifusion kinetics and efficiency were unaffected by the addition of 10-20 mol % BMP to the target membrane. However, using an assay for fusion pore formation and genome exposure, we found full fusion efficiency to be substantially enhanced by the addition of 10-20 mol % BMP to the target membrane, while the kinetics remained unaffected. By comparing BMP to other negatively charged phospholipids, we found the effect on fusion efficiency mainly attributable to headgroup charge, although we also hypothesize a role for BMPs unusual chemical structure. Our results suggest that BMP function as a permissive factor for a wider range of viruses than previously reported. We hypothesize that BMP may be a general co-factor for endosomal entry of enveloped viruses.

biophysics↗

bioRxiv2022

Measuring single-virus fusion kinetics using an assay for nucleic acid exposure

The kinetics by which individual enveloped viruses fuse with membranes provide an important window into viral entry mechanisms. We have developed a real-time assay using fluorescent probes for single-virus genome exposure than can report on stages of viral entry including or subsequent to fusion pore formation and prior to viral genome trafficking. Encapsulating such fluorescence probes in the lumen of target membranes permits specific detection of fusion events and exclusion of leakage without fusion. Using this assay, we show that influenza virus fuses with liposomes of different sizes with indistinguishable kinetics, suggesting that the starting curvature of such liposomes does not control the rate-limiting steps in influenza entry.

biophysics↗