Additional evidence supporting alphavirus entry via a pore at the plasma membrane was obtained using freeze-fracture (Kononchik, Vancini and Brown, Virology, in press). In these experiments, high MOI of SVHR was adsorbed onto mosquito cells for half an hour after which the virus was cross-linked to cell surface proteins with gluteraldehyde. These samples were then processed via a freeze-fracture method to examine the plasma membrane and its components. The technique was a modification of the FRIL (freeze-replicate-immunolabel) technique developed by Fujimoto [87,88]. In brief, the cross-linked samples were flash frozen in liquid ethane, fractured at low temperature and pressure. The fracture plane that goes through the sample follows the path of least resistance, which is the middle of lipid bilayers making two replicas: the inner leaflet, or P-face, and the outer leaflet, the E-face. As a result, when the fracture hits a cell, it tends to follow the plasma membrane. This makes it ideal for examining proteins and interactions that occur on the cell surface. In this experiment, the replicas had cross-linked virus proteins attached to the outside of the E-face. Using immunolabeling, these proteins were labeled, and the organization of the proteins was examined by electron microscopy. The data collected showed labeled virus particles adsorbed to the cells’ surface. These particles were observed as discrete rings of gold bead tags surrounding a structure that traversed the membrane bilayer. We have suggested that this transmembrane structure comprises part of the pore complex.
Summary
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Direct observation of alphavirus-cell interaction by electron microscopy and through the use of chemical inhibitors and interaction with artificial membranes has created seemingly conflicting data. In many direct observations, virus can be seen in endosomes inside the cell [89]. These data suggested that the virus particles enter through an endocytic pathway. Coupled with the conformational changes that occur upon exposure to low pH and the fusion capabilities shown by SINV, the suggested route of entry is similar to that of influenza. However, other observations including thin-sections and freeze-fracture have shown that SINV can infect without endocytosis and acidification. These studies also found that as the particle to PFU ratio of the virus employed improved to unity no virus was seen in endosomes and empty particles were seen on the cell surface. These data suggest that the mechanism for virus entry is at the cell surface and involves direct release of the virus genome into the cytoplasm by a pore-like structure.
The use of chemical inhibitors of endosome formation and acidification has relied on reporter events which occur many steps away from the process of entry. Many of the agents have secondary effect on events that occur after entry which have led to erroneous conclusions.. Likewise studies of virus interaction with artificial membranes have produced a set of requirements for the fusion event that are not related to those occurring with living cells. It appears that, although alphaviruses possess the ability to fuse membranes this is is an event that occurs under special laboratory conditions and may be unrelated to the true entry process.
Arthropod borne viruses are significant sources of disease in man and domestic animals and some are potential agents of bioterrorism. Strategies to control infections by these agents include the development of compounds which will block critical steps in the entry pathway. An accurate image of this pathway and the identification of the cell receptor participating in the entry process is critical for the development of agents which can block infection.