Biology subjects

Villamil Giraldo, A. M.

Publications and source records attributed to Villamil Giraldo, A. M..

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↗

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↗

bioRxiv2019

Detecting and controlling dye effects in single-virus fusion experiments

Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.\n\nStatement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

biophysics↗