Regulation of the Semliki Forest Virus fusion protein by the companion protein

Abstract

The alphavirus Semliki Forest Virus (SFV) infects cells via a low-pH dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with its companion subunit, the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a furin-like cellular protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection.;The processing of p62 was characterized in vivo using a furin-deficient CHO cell line, FD11, and with purified furin in vitro, demonstrating that furin was the authentic p62 processing protease. Unprocessed virus produced in FD11 cells (wt/p62) required a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. Processing of wt/p62 virus particles was observed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection.;To better understand the interaction of E1 and E2 and its impact upon virus fusion, seven p62-cleavage independent (pci) mutant viruses were isolated by growth in FD11 cells. The growth advantage of the mutants in FD11 cells was not due to alternative processing, but due to the fusion of pci/p62 at a substantially higher pH than wt/p62. This higher pH threshold was the result of decreased stability of the p62/E1 dimer in the pci mutants compared with wt p62/E1, allowing formation of the fusogenic E1 homotrimer at a higher pH. Interestingly, the pH dependence of the E2 form of the pci mutants resembled that of the wt even though a less stable E2/E1 dimer was indicated. The impact of pci mutations on fusion of E2 virus was further investigated using the E2 T12I mutation, which causes a lower pH threshold for fusion. A virus containing a pci mutation and T12I showed a higher pH threshold than that of T12I alone.;These studies thus demonstrate that the companion subunit is a critical component of the alphavirus membrane fusion reaction, and suggest important sites for heterodimer interactions that are conserved before and after E2 processing.