Studies on domain III and the stem region of class II viral fusion proteins

Abstract

Alphaviruses and flaviviruses include many serious human and animal pathogens. The viral fusion proteins (E1 and E, respectively) from these two genera have been grouped as "class II" viral fusion proteins, which are distinct from "class I" proteins such as HIV-1 gp41 and influenza virus HA2. Class II proteins consist of 3 domains. Although the domains essentially maintain their original folds during fusion, domain III and the stem (the region connecting domain III to the transmembrane domain) fold back to form a hairpin structure, bringing together the viral and target membranes. Here the alphavirus Semliki Forest virus (SFV) and the flavivirus dengue virus (DV) were used to understand the functions of domain III and the stem of class II proteins.;I expressed, refolded and purified domain III proteins from SFV and DV2. These proteins potently inhibited SFV and dengue virus fusion, and showed cross-inhibition within but not between virus genera. Studies of SFV demonstrated that exogenous domain III bound stably to an E1 trimer intermediate, thereby preventing the folding-back of viral domain III and blocking the initial lipid mixing step of membrane fusion. To study conformational changes of the E1 stem, I generated and purified polyclonal antibodies against specific stem peptides, and showed that the stem packing onto the trimer core in the postfusion conformation prevented antibody binding. Interestingly, this packing was not complete when E1 proteins trimerized without fusion. Stem antibodies and peptides failed to inhibit viral fusion. Sequence comparisons of alphavirus fusion proteins showed several highly conserved residues and a totally conserved length for the stem. Stem mutagenesis revealed important functions of the stem in virus assembly, but showed that neither specific residues nor the conserved length of the stem were required for fusion.;The study with domain III proteins provided the first demonstration of dominant-negative fusion inhibition of class II proteins. The high-affinity and conserved "domain III-trimer core" interaction can serve as an excellent target for developing effective antiviral reagents. The data from domain III proteins, stem antibodies and stem mutagenesis have significantly advanced the understanding of the class II fusion mechanism.