Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E

Envelope protein E of the flavivirus tick-borne encephalitis virus mediates membrane fusion, and the structure of the N-terminal 80% of this 496-amino -acid-long protein has been shown to differ significantly from that of othe r viral fusion proteins. The structure of the carboxy-terminal 20%, the ste m-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane prote in M) during virion assembly, and low-pa-induced structural changes associa ted with the fusion process. To identify specific functional elements in th is region, a series of C-terminal deletion mutants were constructed and the properties of the resulting truncated recombinant E proteins were examined . Full-length E proteins and proteins lacking the second of two predicted t ransmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of so luble homodimeric E proteins. Sites located within a predicted alpha-helica l region of the stem (amino acids 431 to 449) and the first membrane-spanni ng region (amino acids 450 to 472) were found to be important for the stabi lity of the PrM-E heterodimer but not essential for prM-mediated intracellu lar transport and secretion of soluble E proteins. A separate site in the s tem, also corresponding to a predicted alpha-helix (amino acids 401 to 413) , was essential for the conversion of soluble protein E dimers to a homotri meric form upon low-pH treatment, a process resembling the transition to th e fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and virus ass embly.