Interactions involved in pH protection of the alphavirus fusion protein.

The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3-E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

The role of E3 in pH protection during alphavirus assembly and exit.

Alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of E2/E1 glycoprotein heterodimers. During virus entry, the E2/E1 dimer dissociates within the acidic endosomal environment, freeing the E1 protein to mediate fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature E2 and a small peripheral glycoprotein, E3. The immature p62/E1 dimer is acid resistant, but since p62 is cleaved before exit from the acidic secretory pathway, low pH-dependent binding of E3 to the spike complex is believed to prevent premature fusion. Based on analysis of the structure of the Chikungunya virus E3/E2/E1 complex, we hypothesized that interactions of E3 residues Y47 and Y48 with E2 are important in this binding. We then directly tested the in vivo role of E3 in pH protection by alanine substitutions of E3 Y47 and Y48 (Y47/48A) in Semliki Forest virus. The mutant was nonviable and was blocked in E1 transport to the plasma membrane and virus production. Although the Y47/48A mutant initially formed the p62/E1 heterodimer, the dimer dissociated during transport through the secretory pathway. Neutralization of the pH in the secretory pathway successfully rescued dimer association, E1 transport, and infectious particle production. Further mutagenesis identified the critical contact as the cation-π interaction of E3 Y47 with E2. Thus, E3 mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E2 interface.

Cross-inhibition of chikungunya virus fusion and infection by alphavirus E1 domain III proteins.

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (Kd [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.

A key interaction between the alphavirus envelope proteins responsible for initial dimer dissociation during fusion.

Alphaviruses such as Semliki Forest virus (SFV) are enveloped viruses whose surface is covered by an organized lattice composed of trimers of E2-E1 heterodimers. The E1 envelope protein, a class II fusion protein, contains the hydrophobic fusion loop and refolds to drive virus fusion with the endosome membrane. The E2 protein is synthesized as a precursor p62, whose processing by furin primes the heterodimer for dissociation during virus entry. Dissociation of the E2-E1 heterodimer is an essential step during low-pH-triggered fusion, while the dissociation of the immature p62-E1 dimer is relatively pH resistant. Previous structural studies described an "acid-sensitive region" in E2 that becomes disordered at low pH. Within this region, the conserved E2 H170 is in position to form a hydrogen bond with the underlying E1 S57. Here we experimentally tested the role of this interaction in regulating dimer dissociation in mature and immature virus. Alanine substitutions of E1 S57 and E2 H170 destabilized the heterodimer and produced a higher pH threshold for exposure of the E1 fusion loop and for fusion of the immature virus. E1 S57K or S57D mutations were lethal and caused transport and assembly defects that were partially abrogated by neutralization of the exocytic pathway. The lethal phenotype of E1 S57K was rescued by second-site mutations at E2 H170/M171. Together, our results define a key role for the E1 S57-E2 H170 interaction in dimer stability and the pH dependence of fusion and provide evidence for stepwise dissociation of the E2-E1 dimer at low pH.