Molecular Characterization of prM and E Proteins Involved in prM-E Heterodimeric Complex Interaction and Recombinant Subviral Particle Formation of Japanese Encephalitis Virus

• Main Authors: Ying-Ju Lin, 林應如
• Other Authors: Suh-Chin Wu
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/55591743215800040159

博士 === 國立清華大學 === 生命科學系 === 93 === Abstract The formation of flavivirus prM-E complex is an important step for the biogenesis of immature virions, followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. The intracellular formations of the prM-E complexes of Japanese encephalitis virus (JEV) were investigated by baculovirus co-expression of prM and E in trans and prM and E in cis in Sf9 insect cells analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of the carboxyl-terminally truncated prM mutant baculoviruses were constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex. Alanine scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM-binding element for the prM-E heterodimeric stable complex formation. The single amino acid mutation at the His-99 residue of prM abolishing the prM-E interaction was not due to the reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus co-expression of prM and E proteins but not by the prM-H99A mutant. Sequence alignment analysis was further conducted in different groups of flaviviruses to show the prM-H99 residues are generally conserved. The TM regions of prM protein on prM-E heterodimeric complex formation and recombinant subviral particles formation of JEV were further characterized and the TM1 region of prM, especially the specific amino acid sequence-GXXXG motif, was demonstrated using deletion, alanine insertion and glycine substitution mutagenesis. The GXXXG motif located in the TM1 region of prM of JEV were identified for the influence of heterodimerization and virus assembly. Furthermore, the TM regions of E protein on prM-E heterodimeric complex formation and recombinant subviral particles formation of JEV were characterized and the TM1 regions and the conserved charged residues (RDR) of the connecting segment of E were demonstrated using alanine insertion and replacement mutagenesis. These data indicate that the TM regions and His99 of prM and the TM regions and the conserved charged residues (RDR) of the connecting segment of E proteins play a crucial role of the biogenesis of JEV envelope. This information, concerning a molecular framework for the prM and E proteins, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.