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 other viral fusion proteins. The structure of the carboxy-terminal 20%, the stem-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane protein M) during virion assembly, and low-pH-induced structural changes associated with the fusion process. To identify specific functional elements in this 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 transmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of soluble homodimeric E proteins. Sites located within a predicted alpha-helical region of the stem (amino acids 431 to 449) and the first membrane-spanning region (amino acids 450 to 472) were found to be important for the stability of the prM-E heterodimer but not essential for prM-mediated intracellular transport and secretion of soluble E proteins. A separate site in the stem, also corresponding to a predicted alpha-helix (amino acids 401 to 413), was essential for the conversion of soluble protein E dimers to a homotrimeric form upon low-pH treatment, a process resembling the transition to the fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and virus assembly.
Allison
S L
SL
Institute of Virology, University of Vienna, Vienna, Austria. steven.allison@univie.ac.at
Stiasny
K
K
Stadler
K
K
Mandl
C W
CW
Heinz
F X
FX
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus; C065696:glycoprotein E, Flavivirus