Activation of the Alphavirus Spike Protein Is Suppressed by Bound E3

Abstract

Alphaviruses are taken up into the endosome of the cell, where acidic conditions activate the spikes for membrane fusion. This involves dissociation of the three E2-E1 heterodimers of the spike and E1 interaction with the target membrane as a homotrimer. The biosynthesis of the heterodimer as a pH-resistant p62-E1 precursor appeared to solve the problem of premature activation in the late and acidic parts of the biosynthetic transport pathway in the cell. However, p62 cleavage into E2 and E3 by furin occurs before the spike has left the acidic compartments, accentuating the problem. In this work, we used a furin-resistant Semliki Forest virus (SFV) mutant, SFV SQL , to study the role of E3 in spike activation. The cleavage was reconstituted with proteinase K in vitro using free virus or spikes on SFV SQL -infected cells. We found that E3 association with the spikes was pH dependent, requiring acidic conditions, and that the bound E3 suppressed spike activation. This was shown in an in vitro spike activation assay monitoring E1 trimer formation with liposomes and a fusion-from-within assay with infected cells. Furthermore, the wild type, SFV wt , was found to bind significant amounts of E3, especially if produced in dense cultures, which lowered the pH of the culture medium. This E3 also suppressed spike activation. The results suggest that furin-cleaved E3 continues to protect the spike from premature activation in acidic compartments of the cell and that its release in the neutral extracellular space primes the spike for low-pH activation.The alphavirus spike is a trimer of the E1-E2 heterodimer (6,19,42,46). Both subunits are transmembrane glycoproteins (12). E1 carries the membrane fusion function of the virus, while E2 binds the virus to a still ill-defined receptor structure(s) on the cell surface (16,17,32). E2 also controls the fusion function of E1 so that it does not occur before the virus has entered the endosome (23, 38). There, the acidic pH dissociates the heterodimer and allows the E1 to interact with the endosomal membrane via its fusion loop (16,33). This results in E1 homotrimerization and subsequent jack knife like back folding, which brings the viral and the endosomal membranes together for fusion (16,34). The oligomerization of the spike subunits into the heterodimer and the subsequent trimerization of the heterodimers into spikes take place in the rough endoplasmic reticulum of the infected cell (20,25,45). The spikes are then transported to the cell surface via the Golgi complex and apparently also the early endosome (3, 28). At the cell surface, the spikes interact with the viral nucleocapsid and with each other, driving budding of virus particles with T ϭ 4 icosahedral symmetry (6, 10, 31). The fact that the heterodimer is made as an acid-resistant E1-p62 precursor appears to be an elegant solution to avoid premature activation when passing the acidic conditions of the Golgi complex and the early endosome (8, 9, 11, 33). However, the cleavage of the p62 subunit into E2 and the small perip...

The Role of E3 in pH Protection during Alphavirus Assembly and Exit

“…Thus, the change in pH sensitivity between the immature and mature spike proteins appears to be due not to structural rearrangements but to the release of the E3 protein itself. This is in keeping with the finding that E3 remains bound to virus at low pH and is released at neutral pH (19) and provides a possible mechanism to protect E1 during its transit through the secretory pathway. Since dynamic p62-E1 contacts during protein folding in the ER could differ from the static p62-E1 structure, a direct chaperone role for E3 remains unclear.…”

“…The crystal structures of the alphavirus p62/E1 and E2/E1 heterodimers indicate that E3 interacts only with E2 (13,20). Since E3 does not interact with the fusion protein directly, it may act by stabilizing the E2-E1 dimer and thus maintain pH protection until the virus buds from the plasma membrane (19). While this is a compelling model, it has not been tested in vivo in infected cells, and the key interactions within the E3-E2 interface are undefined.…”

“…Following p62 processing, SFV E3 can remain bound to the virus particle at low pH and can be released at neutral pH (19). The bound E3 inhibits virus fusion and E1 trimerization.…”

The Alphavirus E3 Glycoprotein Functions in a Clade-Specific Manner

“…E3 is a small, ϳ65-amino-acid glycoprotein that is dispensable for alphavirus attachment and entry into a new cell (17,37,38,46,51); however, E3 is absolutely required for particle assembly (2,3,23,26). To further investigate a role for E3 in regulating spike assembly, we made and characterized E3 chimeric viruses.…”

“…Once formed, the immature spikes are transported to the plasma membrane through the host secretory system (6). In the trans-Golgi network, the cellular enzyme furin cleaves E3 from E2, which renders the spikes fusogenic (17,37,38,46,51). Lateral spike-spike interactions at the plasma membrane arrange the spikes into hexagonal arrays, which are hypothesized to serve as the sites for E2 and nucleocapsid core interactions to initiate budding from the host cell (44).…”