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

Abstract

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-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. E nveloped viruses use membrane fusion to deliver their genomes into host cells (reviewed in references 1, 2, and 3). These viral fusion reactions can be triggered by various combinations of receptor/coreceptor binding and/or by the low-pH environment of the endocytic pathway. Fusion is mediated by specialized viral fusion proteins, which are highly regulated for deployment at the correct time and place during entry. Suppression of the fusion reaction during virus biogenesis is as crucial as its correct triggering during virus entry. Viruses with low-pH-triggered fusion reactions have evolved several mechanisms to protect their membrane fusion proteins from the low pH of the exocytic pathway. Influenza virus (4) and hepatitis C virus (5) express small membrane proteins, termed viroporins, that act as ion channels and can neutralize the exocytic pathway as a protection mechanism. The alphavirus and flavivirus fusion proteins are synthesized with "companion" proteins whose dimeric interactions protect the fusion protein from low pH (reviewed in reference 6). Maturation of the companion protein by furin cleavage primes the virus to respond to low pH during entry (7,8).Alphaviruses such as Semliki Forest virus (SFV) are plusstrand RNA viruses enveloped in a lipid bilayer containing a lattice of trimeric spikes of E1 and E2 glycoprotein heterodimers (9). The companion protein E2 binds receptors on the cell surface, leading to clathrin-mediated endocytosis...

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion

“…H256(E2) is the only strictly conserved His residue that shows no significant contribution to the two steps investigated in this work. However, previous experimental results suggest that the H-bond network around H256(E2) is important in the pH-dependent regulation of the binding and dissociation of the peripheral glycoprotein E3 (21,23).…”

“…After the virus buds from the cytoplasmic membrane, E3 is released from the virus particle under neutral pH conditions outside the host cell (13,(22)(23)(24).…”

Interactions involved in pH protection of the alphavirus fusion protein

“…We previously used site-directed mutagenesis of the alphavirus Semliki Forest virus (SFV) to destabilize the E3-E2 interface and demonstrate the critical role of E3 in pH protection (Uchime et al, 2013). Our studies identified a conserved tyrosine on E3, Y47, that is required for maintenance of dimer stability during exposure to exocytic low pH, transport of E1 to the plasma membrane, and virus particle production.…”

Cellular and molecular mechanisms of chikungunya pathogenesis

“…Finally, E3 protein is important in directing the structural 117 proteins to the endoplasmic reticulum (ER) for proper assembly 118 and spike formation via its cognate interactions with E2 glycopro-119 tein (Snyder and Mukhopadhyay, 2012). It has also been reported 120 that alphavirus E3 protein could play a role in pH protection during 121 virus formation and budding (Uchime et al, 2013). Upon entry into target cells (Fig.…”