The regulation of pH dependence by alphavirus envelope proteins during virus entry and exit

Abstract

The alphavirus envelope contains two glycoproteins, El and E2, arranged to form an icosahedral shell on the virus surface. El contains the fusion peptide and mediates low pH-dependent fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by the cellular enzyme furin in the trans Golgi network to produce mature E2 and a smaller glycoprotein E3. p62 acts as a chaperone and prevents the premature fusion of El as it is transported through the acidic pH of the secretory pathway.;The first part of my thesis examines a specific interaction involved in destabilizing the El/E2 dimer and thus regulating fusion. Previous structural data described an "acid sensitive region" in E2 that is disordered in the low pH structure. In the center of this region is E2 H170 which is proposed to form a hydrogen bond with El 557, both residues being completely conserved. My work showed that the El 557- E2 H170 interaction has an essential role in the regulation of fusion. We postulate that the initial uncapping of the El fusion loop by E2 domain B occurs via the release of the El 557- E2 H170 interaction, thus affecting dimer stability and the pH dependence of fusion.;The second part of my thesis examines the regulation of the pH dependent fusion protein during virus biogenesis. Since the acid-stable p62/E1 dimer is apparently cleaved before it has exited the low pH compartments of the cell, it is still unknown how El avoids premature activation in these later stages of the secretory pathway. We found that the E3 double mutant Y47A-Y48A was lethal, with only E2 being trafficked to the cell surface. However neutralization of the pH in the secretory pathway successfully rescued dieter association. Subsequent analysis has further shown that the interactions of these two tyrosines with nearby E2 residues are responsible for pH protection during viral exit. My results demonstrated that E3 is required for the pH protection of El during exit from the cell and that the elimination of a single interaction at the E3-E2 interface disrupts this protection.