We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multi step chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the `uncaging' of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid anti-viral anti-fusogenic drug development.
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.
Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-tocell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by Ͼ100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ϳ10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids.IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.
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...
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