The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.The essential initial step in the viral infection process involves transport of the virus or its genetic components through the host cell membrane. Membrane interactions and penetration of viral particles are generally complex multistep processes determined by varied factors and molecular events that have been elucidated for only a few viral species. Indeed, revealing the mechanisms of membrane binding by viral particles, bilayer fusion with the viral coat, and eventual virus internalization into the host cell is critical for understanding viral infection and propagation. In particular, determining the contributions of virus-lipid interactions to cell entry (rather than receptormediated uptake) is essential for gaining insight into the factors affecting cross-species infectivity.Vaccinia virus (VV) is a member of the Poxviridae family, which coinfect a wide variety of mammalian cells. The two infectious forms of vaccinia virions include the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), which are structurally distinct and exhibit different biological features (1). The IMV is a single-enveloped particle containing different viral proteins on its membrane (18) and represents the majority of infectious progeny produced in cells and responsible for virus proliferation among neighboring cells. EEV has an additional outer membrane with specific proteins and corresponds to infecting distant cells (39). The precise mechanisms of VV entry into host cells have not been ful...
The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.
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