Assembly and Entry of Alphaviruses

Sindbis virus, the prototype virus for alphaviruses, is a membrane containing virus with T=4 icosahedral symmetry. It contains two icosahedral shells sandwiching a host-derived lipid bilayer. The outer shell consists of the glycoproteins E1 and E2 and the inner shell is made of capsid and along with the RNA make up the nucleocapsid. The symmetry of the particle has been shown to be directed by the nucleocapsid. Here, data is presented that supports this hypothesis. Using previously generated SV mutants containing furin protease sites at the base of the viral glycoproteins, E1 and E2, the last step of encapsulation can be inhibited. This is hypothesized to be a loss of capsid-glycoprotein interaction at the last critical step of encapsulation. As a result, the virus particles that normally release into the extracellular medium continue to recruit glycoproteins. This creates long tubular protrusions from the plasma membrane that release with agitation. Analysis of the tubular viruses shows assembly consistent with Casper and Klug's hypothesis that simple subunits (E1/E2 trimers in this case) can create icosahedral, tubular and planar symmetry.;Recent crystal structures of soluble constructs of E1 and an E1-E2 fusion protein have been fitted into a cryo-electron microscopy reconstruction giving a pseudo-atomic model of the virus structure. Analysis of this and of these soluble proteins at low pH (6.0) has resulted in a hypothetical conformation of SV at low pH. These data are critically reviewed and highlight unanswered questions and concerns that bring question to their model. An alternative mechanism of entry is also reviewed and compared to the widely accepted mechanism of entry by endocytosis. This mechanism involves infection at the plasma membrane via the use of a proteinaceous pore. Supporting this hypothesis, novel data is presented that show SV cross-linked to a protein complex on the surface of mosquito cells via freeze-fracture replicas. These intramembrane particles (IMPs) are labeled with SV protein antibodies as well as Vacuolar-type H+-ATPase (V-ATPase) antibody suggesting a correlation between the virus receptor and the V-ATPase. In addition to freeze-fracture replicas, thin-sections of SV bound to BHK-21 cells co-label with anti SV and anti-V-ATPase, further supporting the hypothesis that SV and the V-ATPase are associated during infection at the plasma membrane.