With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such as surface plasmon resonance.

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Evaluation of magnetization over time1x10 7 pCT302-TOM22 yeast were magnetized in 2 mL of 50 mM sodium acetate, 50 mM NaCl pH 5 buffer by incubating 100 µL of a 4 mg/mL iron oxide solution for 30 minutes at room temperature. 1x10 7 pCT302-SsoFe2-T2A-TOM22 yeast cells were similarly magnetized, but using 50 mM Tris-HCl, 300 mM NaCl pH 7.4 (TN buffer). Two separate samples were magnetized per cell type. Cells complexed with iron oxide were removed from the unbound cells using a magnet and were washed with the appropriate magnetization buffer. After a single wash, one sample was serially diluted and plated onto SD(-Leu) plates to quantify the initial number of cells magnetized. The other sample was carried forward and washed two further times to simulate the wash and incubation steps that would occur during a typical library selection. After, this sample was incubated with 1% TNBSA pH 7.4. (TN buffer + 1% BSA) for one hour at room temperature. Next, this sample was washed 2X with 0.1% TNBSA pH 7.4 (TN buffer + 0.1% BSA) and incubated for 2 hours at room temperature in this buffer. This sample was finally washed three more times with 0.1% TNBSA, resuspended in 1 mL 0.1% TNBSA, serially diluted, and plated onto SD (-Leu) plates to quantify the cells that remained bound to the iron oxide.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is co-expressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization.

bDetermining how viruses infect new hosts via receptor-binding mechanisms is important for understanding virus emergence. We studied the binding kinetics of canine parvovirus (CPV) variants isolated from raccoons-a newly recognized CPV host-to different carnivore transferrin receptors (TfRs) using single-particle tracking. Our data suggest that CPV may utilize adhesion-strengthening mechanisms during TfR binding and that a single mutation in the viral capsid at VP2 position 300 can profoundly alter receptor binding and infectivity. Canine parvovirus (CPV) is a pathogen of dogs that emerged and caused a pandemic of disease in the 1970s and is Ͼ99% identical in nucleotide sequence to feline panleukopenia virus (FPV), a parvovirus that infects cats and other carnivore hosts but not dogs (1-3). Although the emergence of CPV has been presumed to be the result of a direct transfer of FPV or a similar virus from domestic cats to dogs, we recently demonstrated that CPV exists endemically in sylvatic cycles in North America involving a number of wild carnivore hosts, most notably raccoons (4, 5). These recent findings, along with the lack of isolation or detection of intermediate viruses between FPV and CPV from domestic animals, suggest that parvoviruses transfer frequently between domestic and wild carnivores and that the events preceding the pandemic emergence of CPV were more complex than previously believed (6).Although raccoons have long been known to be susceptible to FPV infection (7), they have only recently been identified as an important host for viruses that are closely related to CPV (4, 8). While CPVs from dogs, wolves, and coyotes all contain a Gly at capsid (VP2) position 300, CPVs from raccoons contain an Asp at that position, suggesting that this mutation is important for the adaptation of CPV to raccoons and possibly other wild carnivore hosts (4, 6). Additionally, VP2 position 300 is the most variable residue in the capsid (9-11). Since FPV and CPV capsids can bind to the transferrin receptor type 1 (TfR), in part by involving the structural region surrounding VP2 position 300 (12), the variations observed at this position appear to be selected by the unique TfR structures of individual carnivore hosts. To examine this phenomenon and to better understand the receptor-binding mechanisms involved, we used single-particle tracking (SPT) techniques to characterize the binding of raccoon-derived CPVs, containing either a 300-Asp or 300-Gly VP2 residue, to dog and raccoon TfRs.The virus studied here was the prototype CPV isolated from raccoons (CPV/Raccoon/VA/118-A/07, GenBank sequence accession number JN867610), which contains an Asp at VP2 position 300 and cannot be propagated in dog cells (4, 6). We refer to this virus as Rac118-300D. However, a single point mutation of the VP2 300-Asp (codon GAT) to a Gly (codon GGT) results in efficient dog cell infec- Citation Lee DW, Allison AB, Bacon KB, Parrish CR, Daniel S. 2016. Single-particle tracking shows that a point mutation in the carnivore parvovirus caps...